PROTEIN CO-CRYSTALLIZATION RECOMBINANT ANTIBODY FRAGMENT
LIBRARY BIOTECHNOLOGY
by
LAURA-LEE CLANCY KELLEY
(Under the Direction of Professor Cory Momany)
ABSTRACT
Co-crystallization recombinant antibody fragments (crFabs) are antibody fragments derived from a parent antibody fragment that can be used to assemble a 3- dimensional matrix (crystal). Our hypotheses were the following:
1. A molecule of known structure can be modified by genetic recombination to draw
another macromolecule along with it into a crystal matrix.
2. The unknown structure can be determined with the aid of the known component.
We have the ability to determine macromolecular structures rapidly. A rate- limiting step is frequently at the crystal formation stage. Typically, only about 20% of macromolecules screened with a large variety of reagents by any available crystallization strategy produce usable crystals. With the development of crFab-based crystallization, those odds may be improved considerably.
Four Fab mutants of a mouse recombinant antibody fragment were tested for their suitability as co-crystallization reagents. The first mutant had a 6-His purification tag on the C-terminus of the heavy chain of the Fab (Fab2). Since Fab2 did not crystallize, the second mutant was modified to have four vicinal histidines, two on each chain. It was
discovered that this mutant had a leucine residue inserted into a beta-pleated sheet when compared with mFab25.3. Fab4 was then constructed by a deletion mutation of that residue. Finally a heavy chain (N57→K) mutation was created to engineer a stronger salt
bridge between the Fab molecules as they packed into the crystal structure (Fab5).
Mutant rFab4 proved to be the best mutant for co-crystallization studies based on its yield
and crystallization properties. By multi-site directed mutagenesis of the third
hypervariable regions of the light and heavy chains, a recombinant Fab antibody
fragment library was built with a nominal diversity of 1.16 x 107 colony forming units.
This library was shown to produce rFabs that could recognize seven out of seven proteins tested and has the potential for being used for the co-crystallization of proteins.
INDEX WORDS: Antibody fragment library, Multi-site directed mutagenesis,
Biotechnology, Crystallization
CO-CRYSTALLIZATION RECOMBINANT MOUSE ANTIBODY FRAGMENT
LIBRARY BIOTECHNOLOGY
by
LAURA-LEE CLANCY KELLEY
B.S., Michigan State University, 1972
A. A. S. Nursing, Community College of Allegheny County, 1982
M.S., University of Pittsburgh, 1986
A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial
Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
ATHENS, GEORGIA
2003
© 2003
Laura-Lee Clancy Kelley
All Rights Reserved
CO-CRYSTALLIZATION RECOMBINANT MOUSE ANTIBODY FRAGMENT
LIBRARY BIOTECHNOLOGY
by
LAURA-LEE CLANCY KELLEY
Major Professor: Cory Momany
Committee: Bi Cheng Wang E. W. Taylor N. G. Newton A. Capomacchia Mark Eiteman
Electronic Version Approved:
Maureen Grasso Dean of the Graduate School The University of Georgia May 2003
DEDICATION
I would like to dedicate this dissertation to my husband, David D. Kelley, for his constant encouragement, supportiveness and faithful devotion. Also, it is dedicated to the memory of my father who was proud to have me for his daughter and encouraged all of his children to follow after whatever God put in their hearts to do.
iv
ACKNOWLEDGEMENTS
I would like to acknowledge those who have helped me with my academic career.
My high school physics teacher, Mr. Ralph Facziska, made physics understandable, interesting and practical, by showing me a side of science that was the exact opposite of the attitudes expressed by most of my peers. Also, I will be forever grateful to the Dean of the Faculty of Arts and Sciences at the University of Pittsburgh, Jerald Rosenberg, for
his encouragement while I was an undergraduate student and his friendship over the years
and to Professor John Rosenberg for introducing me to the science of crystallography.
Professor Bi-Cheng Wang, affectionately known as “B.C.” to all his students and
colleagues, has been a source of inspiration by promoting my career as a student here at
the University of Georgia as well as having been the chairman of my master’s committee
at the University of Pittsburgh. He is an excellent teacher of protein crystallography. His
inclusion of us at his parties has been helpful in many ways.
Professor Cory Momany has been a wonderful source of advice and an excellent
director of the progress I have made in my research on the topic discussed in this
dissertation and for his instruction in the use and maintenance of laboratory equipment
that was new to me. He has willingly been “on call” for late evening questions regarding
instrument problems every time they arose. I have enjoyed working with him for the past
four and a half years.
Professor Anthony Capomacchia has truly been a student advocate for me as well
as others.
v
Professor E. William Taylor has graciously allowed me to use his PCR equipment
and has also spoken encouragingly to me at times as well as contributing to committee
meetings in such a way that made me feel he was on my team.
Professor Emeritus Gary Newton has also provided insightful comments. I have
appreciated having him on my committee. Every member of the committee has been
wonderful to me and a great group to know.
Professor Mark Eiteman in the Department of Biological & Agricultural
Engineering provided very useful conversations on bioreactor processes and graciously allowed me to use his facilities. I appreciate his willingness to join the committee at this late hour. Geoffrey Smith and Sarah Lee were also helpful in Dr. Eiteman’s lab for assisting to set up the bioreactor experiments. Atin Tomar was particularly helpful when he took the late night samples on the second bioreactor process.
I would also like to express my appreciation of the people I have worked along side of in the Momany lab in the last four years: the Russian post-doctoral fellows Elena
Blagova and Vladimir Levdikov, especially for helping me with the HPLC instrument and their rallying support when I needed help to move from one apartment to another, and fellow graduate students, Nandita Bose, Ph. D., Alex Vandell, Pharm. D., and Jay
Houston, for their encouragement and help both professionally and personally. Various undergraduate students have contributed to the diversity of the lab, but Betty Ngo was an outstanding technician and Avis Scott shows great potential as a lab worker.
vi
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ...... v
LIST OF TABLES ...... x
LIST OF FIGURES ...... xi
CHAPTER
1 BACKGROUND AND SIGNIFICANCE...... 1
1.1 Overview...... 1
1.2 Crystallization of Proteins ...... 2
1.3 Antibodies, Types of Antibody Fragments and their Structure ...... 6
1.4 A Brief History of Biotechnology and Antibodies ...... 10
1.5 Phage Display Mechanism in Escherichia coli...... 12
1.6 Significance ...... 15
2 THE CURRENT STATE OF THE ART OF RECOMBINANT
ANTIBODY FRAGMENT BIOTECHNOLOGY...... 17
2.1 Overview...... 17
2.2 Problems Associated with Recombinant Antibody Fragment
Biotechnology...... 17
2.3 Applications of Antibody Fragment Biotechnology...... 23
2.4 Future Applications and Developments in Biotechnology...... 33
2.5 Summary ...... 37
vii
3 OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION
OF MOUSE RECOMBINANT ANTIBODY FRAGMENTS IN
ESCHERICHIA COLI...... 38
3.1 Overview...... 38
3.2 Rationale...... 39
3.3 Materials and Methods ...... 40
3.4 Results...... 51
3.5 Discussion...... 74
4 MOUSE RECOMBINANT ANTIBODY FRAGMENT PRODUCTION
IN A BIOREACTOR ...... 76
4.1 Overview...... 76
4.2 Rationale...... 76
4.3 Materials and Methods ...... 77
4.4 Results...... 81
4.5 Discussion...... 87
5 GENERATION OF A PHAGEMID MOUSE RECOMBINANT
ANTIBODY FRAGMENT LIBRARY BY MULTI-SITE DIRECTED
MUTAGENESIS ...... 89
5.1 Overview...... 89
5.2 Rationale...... 90
5.3 Materials and Methods ...... 91
5.4 Results...... 99
viii
5.5 Discussion...... 114
6 CONCLUSIONS AND FUTURE DIRECTIONS...... 116
6.1 Conclusions...... 116
6.2 Future Production and Development of the crFab Co-Crystallization
Library ...... 118
REFERENCES...... 120
APPENDICES
A LIST OF ABBREVIATIONS ...... 140
B SHORT PROTOCOLS ...... 142
C pET28 FABN DATA...... 147
D PHAGEMID GENEBANK ACQUISITION DATA ...... 154
ix
LIST OF TABLES
Page
Table 3.1: Primer Sequences for PCR Mutations to pET28 Plasmids...... 43
Table 3.2: Design of the Diaminopimelate/IPTG Experiment...... 50
Table 3.3: Physical Characteristics of rFab Mutants...... 63
Table 4.1: First Bioreactor Fermentation Process O. D. 600 nm, % DO, Acid and
Base Consumption over Time...... 83
Table 4.2: Second Bioreactor Fermentation Process O. D. 600 nm, % DO, Acid and
Base Consumption over Time...... 86
Table 5.1: Sequence Primers Used for pCOMB-Fab4 PCR Amplification...... 93
Table 5.2: PCR Parameters for the crFab Library Using the Multi-Site Directed
Mutagenesis Kit...... 96
Table 5.3: Electroporation Results for pCOMB-Fab4 Transformation in E. coli.....99
Table 5.4: DNA Results for the 12 pCOMB-Fab4 Transformed Colonies...... 100
Table 5.5: Colony Count Results from Preliminary PCR Transformations...... 105
Table 5.6: Colony Count Results for Library Panning...... 110
Table 5.6: ELISA Results for Library Panning of Seven Proteins...... 111
x
LIST OF FIGURES
Page
Figure 1.1: Successful Crystallization Methods Based on the Percentage of
Crystallized Proteins...... 3
Figure 1.2: Antibody Structure and Substructures...... 8
Figure 1.3: Structural Organization of Artificial Antibody Fragments...... 11
Figure 1.4: Schematic Drawing of a Filamentous Phage...... 14
Figure 2.1: Multi-Step ELISA Procedure versus a Single-Step OS-BLIA
Procedure ...... 24
Figure 3.1: Protein Induction and Purification Process...... 45
Figure 3.2: Insertion of the “fab” Genes into the pET28 Vector...... 52
Figure 3.3: Recombinant Mutants of the Heavy Chain...... 54
Figure 3.4: Recombinant Mutants of the Light Chain ...... 55
Figure 3.5: HPLC rFab3 Purification Results for Defined Medium Versus
Periplasm ...... 58
Figure 3.6: Q Column Purification Results for rFab3 from Periplasm and
Supernatant Fractions from the Same 1-Liter batch...... 59
Figure 3.7: Comparison of HPLC Purification for rFab4 and rFab5 ...... 60
Figure 3.8: HPLC Results for T-gel Purification of rFab4 from Defined Medium...62
Figure 3.9: Native Gel of rFab Proteins with p24...... 64
Figure 3.10: Purification of rFab4 from Periplasm and Defined Medium...... 66
xi
Figure 3.11: High Density SDS-PAGE Gel of rFab4 Mutant ...... 67
Figure 3.12: Crystals of rFab3, rFab4 and rFab5 Proteins ...... 69
Figure 3.13: Diaminopimelate-IPTG Induction of pET28-Fab3...... 71
Figure 3.14: Growth of E. coli and Production of rFab Mutants ...... 73
Figure 4.1: Bioreactor Process Utilizing On-Line Glucose Feeding...... 79
Figure 4.2: First rFab4 Bioreactor Process ...... 82
Figure 4.3: Second rFab4 Bioreactor Process ...... 85
Figure 5.1: Relative ELISA Response of 12 pCOMB-Fab4 Clones ...... 102
Figure 5.2: pCOMB Phagemid Vector Containing the Light (fabL) and Heavy (fabH)
Sequence Data ...... 103
Figure 5.3: Panning Cycle Results for the Seven Proteins...... 108
Figure 5.4: Agarose Gels of Restriction Endonuclease Cuts...... 113
xii
CHAPTER 1
BACKGROUND AND SIGNIFICANCE
1.1 Overview
This chapter covers the crystallization of proteins, briefly how they are crystallized, what one of the problems of protein crystallization is, our hypotheses and our solution to the problem of macromolecular crystallization. Then information on antibody co-crystallization using an antibody fragment as a self assembling matrix or as a co-crystallization reagent will be presented. A background on antibody structure and terminology will be provided including a discussion of the general immunological uses of antibodies and specific uses of antibody fragments. The types and structures of antibodies, in general, and antibody fragments of various immunological types will be described and illustrated in reference to their uses. A brief overview of biotechnology follows with significant events noted. The phage life cycle with infection of Escherichia coli and phage display of proteins will be described. The production of phagemid antibody fragments with selection of targets by panning will be discussed briefly in
relation to the subject of this dissertation. Finally, the significance of this project will be
highlighted.
1
1.2 Crystallization of Proteins
Two methods for determining the 3-dimensional structure of molecules at a detailed level are currently in use, x-ray crystallography and nuclear magnetic resonance
(NMR). Crystal structures offer a static view with detailed insight into both structure and function. Numerous functional implications are theorized from the snapshot that three- dimensional x-ray crystallography gives us of these structures. For example, multiple static views of the same enzyme under different conditions of crystallization, with and without substrates and/or cofactors, have led to a detailed understanding of mechanisms of action for many enzymes. NMR structures provide insight into the flexibility of these molecules in solution, but there is a size limitation of about 30kDa.
Crystallization is an empirical process. There are numerous ways to produce crystals but currently the most popular are hanging-drop or sitting-drop vapor diffusion and micro-batch methods. The variables can include pH, temperature, salts, buffers, additives, precipitants, co-factors, heavy atoms and detergents. A graph illustrating the kinds of methods and their relative popularity based on the percentages of total proteins crystallized is shown in Figure 1.1 (Cudney & Patel, 1994). The bulk crystallization method is less popular because it requires milligram quantities of the protein and many proteins are too scarce to squander on a technique that may not work. Seeding is usually used when only tiny crystals less that 0.2 mm can be obtained by other methods. Free interface diffusion requires the layering of the protein solution atop of the precipitant solution in a glass capillary tube with the ends of the tube sealed in some manner to prevent drying. The glass capillaries are very fragile and layering the solutions with a
Hamilton syringe is tricky so that there is no air space between the two. Dialysis works
2
well when one has a fairly substantial amount of protein. It can be used to change crystallization conditions for the same sample over time and as a concentration method that will occasionally produce crystals by accident.
Bulk Crystallization Batch Method Dialysis Seeding Free Interface Diffusion Vapor Diffusion Temperature Induced
Figure 1.1: Successful Crystallization Methods Based on the Percentage of
Crystallized Proteins (Cudney & Patel, 1994.)
We now have the ability to determine macromolecular crystal structures rapidly with recent technological advances. The rate-limiting step after a protein is obtained and purified is at the crystal formation stage. Typically, ~20% of macromolecules screened with a large variety of reagents produce crystals by any available crystallization strategy
(G. DeTita, Hauptman-Woodward Institute, Buffalo, NY, private communication). With the development of a co-crystallization Fab (crFab) antibody fragment-based
3
crystallization and possibly with the aid of robotics, we hope to improve those odds considerably.
Implicit in the use of crFabs in crystallization are two hypotheses:
1.A preexistent molecule of known structure can be modified by genetic
recombination to draw another macromolecule along with it into a crystal
matrix.
2.The unknown structure can be determined with the aid of the known
component.
Structure determinations using molecular replacement have now become routine provided that there is minimal structural rearrangement of the selected protein’s main chain. Relative to the model, the remainder of the structure can be deduced through an iterative process once the molecule has been properly oriented within the crystal lattice.
A Self-Assembling Matrix (SAM) is a group of objects that assemble themselves into a 3-dimensional matrix (crystal). A SAMFab is a genetically engineered antibody fragment that is designed to draw its cognate antigen along with it into a crystal having the same crystal space group with identical cell constants regardless of the cognate antigen (target molecule) as its parent monoclonal Fab. While this dissertation project was begun with the goal of creating a SAMFab library the goal was not met because of the non identical space group of the recombinant antibody fragment and difficulties in engineering the antibody fragment itself.
Co-crystallization recombinant antibody fragments (crFabs) are antibody fragments that have been genetically engineered as co-crystallization reagents. In our implementation, poly histidine purification tags were added to facilitate their purification.
4
The crFabs may or may not crystallize in the identical space group and have the same cell constants. One of the obvious differences between crFabs and SAMFabs is that the volume occupied by the target in the crystal lattice may vary considerably from crystal to crystal.
Hydrogen-bonding networks may be different from crystal to crystal in crFabs, based on specificities of the mutated side chains.
Proteins are not the only targets for detailed structural analyses. DNA, RNA and carbohydrates are also important biologically. Sugar moieties on the surface of organisms like Diplococcus pneumoniae (Streptococcus pneumoniae) may protect them from host recognition by the immune system. However solving their 3-D structures is problematic. Surface carbohydrates on proteins are quite mobile. If their flexibility can be minimized by a systematic interaction with a specific antibody fragment to form a crystal, then they too can be studied by x-ray analysis.
Membrane proteins have posed a special problem because of the “inside out” nature of their structures. Instead of having a hydrophobic core surrounded by hydrophilic surface residues, the opposite is true. Membranes are hydrophobic and proteins imbedded in them have to have a hydrophobic outer structure and frequently have a hydrophilic core if they act as transport molecules. Hydrophobic proteins must be solubilized to be crystallized. Special detergents like β-octyl-D-glucopyranoside (utilized to help crystallize prostaglandin H synthase) have been developed to aid with this problem but antibody fragments can be developed to interact with membrane proteins as well.
5
1.3 Antibodies, Types of Antibody Fragments and their Structure
Historically, immunity meant protection from infectious diseases. In 1900 Paul
Ehrlich provided a theoretical framework for the antigen–antibody interaction that was designated the “humoral theory of immunity.” Since that time a great deal has been learned about antibodies and their specificity. The portions of foreign proteins, carbohydrates or polysaccharides that are antigenic are called epitopes or determinants.
Antibodies are produced in mammalian systems by specialized cells called B lymphocytes (derived from the bone marrow but originally thought of as derived from bursa cells). The fundamental concept of the clonal selection hypothesis is that “every individual possesses numerous clonally derived lymphocytes, each having arisen from a single precursor and being capable of recognizing and responding to a distinct antigenic determinant, and when an antigen enters, it selects a specific preexisting clone and activates it.” The start of a clone is a single cell that divides in response to the introduction of an antigen. Monoclonal antibodies are specialized antibodies that the mammal produces in response to repeated exposure to specific antigens. The technique of producing monoclonal antibodies outside the mammalian body requires the fusion of a
B cell with a myeloma cancer cell in what then becomes a hybridoma cell line that immortalizes the monoclonal B cell. Hybridoma cells also produce monoclonal antibodies (Abbas et al., 2000, #1). Polyclonal antibodies are antibodies produced by more than one clone against different epitopes of the same antigen.
There are five types of antibodies, IgA, IgD, IgE, IgG and IgM, depending on their heavy chain. IgG, IgD and IgE are monomers. IgA is a trimer linked by J chains and IgM is a pentamer also linked by J chains at the base of the tail portion. There are
6
five types of heavy chains (α, δ, ε, γ or µ). There are only two types of light chains in mammals, either κ or λ, and they can associate with any of the five heavy chains. See
Figure 1.2 for a schematic diagram of the intact immunoglobulin and its substructures.
Two light chains associate with two heavy chains to form the basic monomeric Y- structure. Antibody fragments have been generated in the past by one of two methods, either papain cleavage (2 Fabs plus 1 Fc) (Porter, 1959) or pepsin digestion (Fab’2).
Pepsin digestion removes the Fc portion, leaving the hinge regions and the two “arms” intact with their variable regions. Papain cleavage produces two usable Fab fragments and an Fc fragment (Abbas et al., 2000, #2).
The canonical antibody structure consists of a β-barrel called the immunoglobulin fold. The immunoglobulin fold consists of repeats of antiparallel β-pleated sheets, turns and loops (Chang et al., 1985). The β-pleated sheets are stacked on each other in their respective domains primarily by main-chain hydrogen bonding and disulfide bridging
(Edmundson et al., 1993, Edmundson et al., 1996, and Harris et al., 1998). Van der
Waals forces and pi stacking of phenyl, tryptophan and tyrosine rings may also play important roles in individual interactions and in forming the hydrophobic core of these proteins. Salt-bridging and hydrogen bonding between side chains are more important in target recognition. In the complete immunoglobulin (Ig) the four monomers (2 heavy and
2 light chains) intertwine (β-pleated sheet-stacking causes a left-handed twist) forming a
Y structure that has two equal “arms” (Fabs) and a “tail” that is called the constant region
(Fc). Each of the “arms” (Fab) has a variable region that interacts directly with the target molecule (antigen) and a structurally conserved constant domain. The “tail” (Fc) portion, consisting of two or more structural domains, interacts with specific cell receptors or is
7
Fab2’
H Heavy chain C D R 1 H C D R 2 Light chain H C D R L 3 C D R 1 L C D R papain 2 L C D R cleavage 3
VL - - S S -S - - -S Fab
CL -S-S- pepsin digestion
Fc
carbohydrate
Figure 1.2: Antibody structure and substructures. There are two heavy and two light chains disulfide-bridged and hydrogen-bonded together to form a multi-domain structure. Abbreviations used are Fab, antibody fragment derived from papain cleavage; Fab2’, bivalent pepsin antibody fragment derived from pepsin digestion;
CDR, complementarity determining region; VL, variable domain of the light chain; CL, constant domain of the light chain; Fc, heavy chain constant domain tail fragment.
Papain cleavage (2 Fabs plus Fc). and pepsin digestion (Fab’2) points noted. The highly flexible hinge regions are shown as thin segments of the heavy chains between the Fc and the Fab CL domains where papain cleavage occurs. Each chain has three
CDR loops, HCDR 1-3 and LCDR 1-3.
8
linked by J chains to identical Y structures in the larger antibodies, IgA and IgM. For a more detailed description of canonical antibody secondary structures refer to Chothia &
Lesk, 1987, Al-Lazikani et al. (1997 & 2000) and Morea et al., 1998.
Flexibility in the structure comes about in two ways. There is an “elbow” between the Fab heavy constant and variable domains that gives it several more degrees of freedom in interacting with the target molecule and may be required for high-affinity binding with the target (Landolfi et al., 2001). Between the Fab portion and the Fc region is a hinge region on the heavy chain that provides extreme flexibility in this region. (Abbas et al., 2000, #2.)
The domain structure of the Fab is such that in the Variable Region there are β- pleated sheets that are relatively constant in structure the “framework” regions and highly variable loops called the “complementarity determining regions” (CDR) or hypervariable loops connecting the β-pleated sheets (Chothia & Lesk, 1987). These CDR loops are important for specific interactions with the epitope of the antigen. In the Fab Constant
Region there are also β sheets and β turns between the sheets that do not interact directly with target molecules but play structural roles in packing the sheets together and may have positive stacking or ribbon-forming interactions with other β-pleated sheets in the packing of Fabs in crystals. The specificity and variability of Fab molecules and their ease of crystallization make them attractive as molecules for co-crystallization (Kovari et al., 1995). Figure 1.3 demonstrates schematically the different designs of smaller antibody fragments called Fv for “fragment variable” domain. Monoclonal antibody production is limited by its dependence on a mammalian source to produce the desired antibodies. On the other hand, a recombinant protein expression system allows for
9
genetic manipulation of the product and has other advantages. Recombinant antibody
fragments (rFabs) have a much greater potential as aids to structure determination, as
laboratory tools, and for diagnostic and therapeutic applications because of their ease of
production and the greater degree of control the bioengineer can exercise over the final
product. If glycosylation of the product is not an issue, then prokaryotic production can
be performed cheaply.
1.4 A Brief History of Biotechnology and Antibodies
Biotechnology as a term was not recognized until about 1977. Many events had to take place before biotechnology could exist. For example, curiosity about things very small led to the development of the microscope (1590) which eventually brought the substructures of cells into view and a desire to know those structures and their functions in detail. Fermentation to produce beer had been discovered by the Sumerians c. 1750
B.C. but isolating and culturing bacteria as we do today did not come about until well after their discovery in 1675 A.D. Without the discovery of E. coli in 1855 and its subsequent utilization as a labtool, much of the following research could not have taken place. The discovery of x-rays in 1895 eventually led to our use of x-rays to determine macromolecular structure. Both the developments in molecular biology since the late
1930’s and more recent court cases legitimizing the use of recombinant technology have been essential to the growth of biotechnology. For antibody fragment biotechnology to
exist today, we must take a closer look at the progress in understanding antibodies in
general. Humoral immunity is mediated by proteins in the blood called antibodies. In
the mid 1950’s a hypothesis of clonal antibody production was put forth by Jerne (1955)
and modified by Burnet (1957 & 1976). Jerne
10
a.A Bb. LCDR1 LCDR2 LCDR3 LCDR1 LCDR3 LCDR2 HCDR2 HCDR3 HCDR1 HCDR2 HCDR3 HCDR1
cC. Dd. LCDR1 LCDR2 LCDR3 LCDR2 LCDR1 LCDR3
TOXIN HCDR3 HCDR1 HCDR2 HCDR2 HCDR1 HCDR3
Figure 1.3: Structural Organization of Artificial Antibody Fragments. Panel A an
Fv, panel B an scFv without a linker, panel C an scFv with a 15 residue linker of glycine and serine residues and panel D an scFv with a 15 residue linker and a fusion protein
(usually a chimeric molecule). Hydrogen bonding usually occurs between the main chain
β-sheets in the framework regions between the heavy and light chain segments that are pictured above as either yellow or orange segments. The green and blue boxes represent the variable loop regions that are more likely to hydrogen- bond and/or salt-bridge through their side chain atoms with their cognate antigen (target molecule).
Abbreviations used are: LCDR1-3, light chain complementarity determining regions 1-3;
HCDR1-3, heavy chain complementarity determining regions 1-3.
11
(1974 & 1982) also hypothesized that both foreign antigens and self structures can be
mimicked by our own immunoglobulins to produce anti-idiotypic antibodies. This was
demonstrated recently with the aid of a human Fab phage-displayed library (Vogel et al,
2000). The specificity and high affinity of antibodies for their cognate antigens were demonstrated. A means of immortalizing B lymphocyte clones was discovered in 1975
(Kohler & Milstein). Since that time monoclonal antibodies (Mabs) have been produced by a combination of immunization and hybridoma technology. For utility Mabs have been further reduced to either one of two antibody fragment types by either pepsin cleavage (two Fab plus one Fc) (Porter, 1959) or papain digestion (Fab2’)
Monoclonal antibody fragments (mFabs) have been used to help derive structures
of other proteins. The elegant structures of Pokkuluri et al., 1994, Momany et al., 1996,
and of Ding et al., 1998, provide us with a few examples of how these Fabs have served
us as crystallization vehicles.
Heterodimeric recombinant Fab fragments have been displayed on the surface of
a phage (Love et al., 1989) and in E. coli preparations of periplasm fractions as well as in
various fermentation media. The in vitro production of a single-chain variable fragment
(scFv) became feasible in the early 1990’s (McCafferty et al., 1990, Marks et al., 1991).
ScFv’s have also been used to co-crystallize proteins with cytochrome c oxidase (Essen et al., 2003) being a clear example.
1.5 Phage Display Mechanism in Escherichia coli
Filamentous phages f1, fd and M13 have been intensively studied (reviewed by
Smith, 1985.) The phage genome consists of ten protein coding genes, one of which
12
codes for the phage major coat protein (Gene VIII) and another codes for the few copies
of pIII that are necessary for attachment to the F pilus on E. coli and adsorption into the
cell. Protein III is essential for infectivity of the phage particles. These phages are very
similar to one another and the M13 phage has been further adapted for laboratory use by separating out portions of its genome to produce a phagemid that requires a helper phage
VCSM13. The use of helper phage increases the control the researcher has over the
phage display process. Figure 1.4 is a simplified drawing of a filamentous phage with its super-coiled single-stranded circular DNA core. The axial ratio is actually much longer than shown here, about 140 to 330 times longer than it is wide (Model & Russel, 1988).
The life cycle of the phage in E. coli begins with attachment of the phage to the F pilus of
F+ E. coli. The F pilus then retracts into the bacterium by reabsorbing the molecules that
make up the pilus until the attached particle is also absorbed (in this case, the phage).
Once inside the bacterium, the phage protein coat disassembles and the single-stranded
DNA (ssDNA) is released. The host enzymes make double-stranded DNA (dsDNA)
using the single-stranded DNA as a template. This dsDNA is the replicative form of the
phage. The host enzymes are then subverted into producing the 10 proteins encoded by
the phage dsDNA in the cytosol. The protein coat and plus strand of the ssDNA are
assembled in the periplasmic space of the E. coli and phage are then shed into the
medium to begin the cycle of infection of other F+ E. coli bacteria all over again.
By expressing a protein in frame with the phage’s surface protein, one can link
genotype to phenotype. This led to the concept of phage display of peptides and proteins
on the surface and the method of selection called panning. Panning is the process
whereby a phage displaying a protein (Fab, scFv) is incubated with an immobilized target
13
pVIII ssDNA pIII
Figure 1.4: Schematic Drawing of a Filamentous Phage. The outer coat consists of a
major coat protein pVIII that surrounds the super coiled single-stranded DNA core and
the minor protein pIII that is a used for attachment to the F pilus of F+ E. coli.
molecule (antigen), allowed to bind and the bound phage eluted with low pH, antigen or trypsin. Then the phages with high affinity for the molecule of interest are re-amplified in
Escherichia coli.
The use of phage display of peptides and proteins on the surface of phage particles has brought about an immunization-free and hybridoma-free method of producing antibody fragments (Smith, 1985, Model & Russel, 1988, Burton & Barbas,
1993, Burton, 1995 and de Haard et al., 1999). This discovery led to the production of single chain variable region fragments (scFv) that incorporate only the variable domains
of both the heavy and light chains fused together with or without a (Gly4Ser)3-peptide
linker. It has since been demonstrated that the larger antibody fragment of independent
14
heavy and light chains will self-associate to form heterodimers in solution or on the
surface of the phage (Love et al., 1989). However, Reiter et al., 1999, also have demonstrated that even the simplest single domain of a heavy chain variable region is useful for targeted therapy and imaging purposes.
The simple prokaryotic E. coli organisms with their phage parasites offer many advantages, such as simple culture media, rapid growth rate and simpler extraction from the cells or growth media, over mammalian cell culture. Maintenance and storage facilities for DNA and for prokaryotic cell lines are considerably reduced when compared with housing and maintaining animals or hybridoma cell lines. The filamentous phage supply a uniquely simple system of phage display of a peptide or Fab on their minor coat protein with the corresponding DNA and only infect one E. coli cell per phage (Smith,
1985, Reiter et al., 1999). They do not require large amounts of storage space or complicated feeding and housing protocols for animal maintenance. Also, once cloned, the DNA is much more amenable to manipulation.
1.6 Significance
The purpose in producing, purifying and crystallizing proteins is relevant to today’s search for a depth of understanding of and intervention in many disease processes. The development of pharmaceutical agents effective in numerous diseases is often dependent on a precise knowledge of the structure of the enzyme or receptor.
However, only a few of the key drug metabolizing membrane proteins, e.g. cytochrome
P450 monooxygenases have been characterized in molecular detail (Cupp-Vickery et al.,
2000, Hubbard et al., 2001, Williams et al., 1997 & Zhao et al., 1999). Very few
15
membrane protein structures have been solved because of the inherent difficulties associated with these lipid soluble proteins.
The objectives of producing a library of Co-Crystallization Antibody Fragments are to find clones to specific targets (proteins, complex carbohydrates, small molecules, etc.). Amplification of the crFabs to the specific targets will produce enough material to use in co-crystallization trials. We have a large database of crystallization conditions to draw from now because of a cumulative 50 + years of experience at crystallizing proteins. At least theoretically, this will in turn produce usable crystals of these previously un-crystallized proteins and other molecules that will lead to their 3- dimensional molecular structure.
16
CHAPTER 2
THE CURRENT STATE OF THE ART OF RECOMBINANT ANTIBODY
FRAGMENT BIOTECHNOLOGY
2.1 Overview
Current literature about the biotechnology of antibody fragments is reviewed in this chapter. There have been problems associated with phage display, but innovations have frequently followed those problems. The problems and their solutions will be discussed. The topic of the second half of this chapter is a diversity of applications that have been found for antibody fragments. Looking at what has already been done leads us to look at possible applications and developments in the near future.
2.2 Problems Associated with Recombinant Antibody Fragment Biotechnology
Since any new technology encounters difficulties, phage display is no exception.
Existing phage display vectors have several problems and drawbacks. Some antibody fragment libraries are created by starting with two separate vectors, one for producing the heavy chain and another for producing the light chain. These are separately mutagenized by degenerate primers. They recombine these via PCR of the gene products into a group of vector products en mass and then transform the recombined antibody fragments into E. coli cells. There is no direct means of identifying non-recombinant clones unless the
17
recombinant clone is restructured in some way that makes it visually different from the
non-recombinant clone (selection by chloramphenicol or visual inspection of a green
fluorescent tag). Phage elution during the panning cycles is required for amplification of recombinant antibody phages of interest. There are no specific protease cleavage sites for proteolytic cleavage of proteins from the surface of phages. Trypsin and other commonly used proteases are too general and degrade the protein of interest. An alternative may be to add a chitin-binding site that can be cleaved readily (Tsujibo et al., 2000 & Blank et
al., 2002).
Leaky promoters (Zahn et al., 1999) and restriction sites that are incompatible
with the clone of interest have been problematic. New vectors are being developed to
improve phage display as well as to improve large-scale production of proteins (Matthey
et al., 1999, Paschke et al., 2001 & Zahra et al., 1999). One of the newer vectors (pGP-
F100 based on vector pGZ1) features a laco/p-GFPuv stuffer fragment for easy detection
of clones carrying the non-recombinant phagemid. GFPuv is the mutant of Agueoria victoria green fluorescent protein that was optimized for growth in E. coli at 37ºC.
Transformed colonies carrying the vector are visually identified by their intense green
color. An innovation in restriction sites is insertion of a Tev protease heptapeptide
recognition and cleavage sequence into the amino acid sequence of the widely used myc
tag (MyCut tag). With the addition of Tev protease this allows rapid removal of about
90% of the phage bound to the target in the panning phase (Paschke et al., 2001). A
trypsin recognition amino acid sequence of KDIR can also be used.
The yield of antibody fragments recovered from a fermentation process is
dependent on two factors, high-level expression and degradation due to the presence of
18
intrinsic E. coli proteases. Low protease strains of E. coli are available commercially, but
it is not possible to produce viable strains with no proteases. A protein expressed at high
levels that is readily attacked by intrinsic proteases will produce a low yield of
recombinant protein. The yield of functional recombinant antibodies is often very low
(Krebber et al.,1997). Single amino acid residue changes can significantly change the amount of the antibody fragment recovered especially if the location of that amino acid residue makes that segment more susceptible to E. coli protease degradation (de Haard et
al., 1998). The yield of antibody fragments in a particular strain of E. coli can even be
dependent on unusual culture conditions such as the addition of 0.4 M sucrose
(Kipriyanov et al., 1997).
One of the difficulties with the Fab type of recombinant antibody fragments is that
the product must be a heterodimer of a light chain and a heavy chain. In solution it is
possible to get heavy chain homodimers and light chain homodimers (Bence Jones
protein) if there is no selection pressure for the heterodimer. Early work with attempts to
produce recombinant heterodimers of antibody fragments lead to patchwork-quilt type of
molecules because of the seeming necessity to engineer purification handles into one or both polypeptide chains. Atwell et al., 1997, have approached the problem of identifying recombinant clones by remodeling the domain interface of a homodimer into a heterodimer by using site-directed mutagenesis of targeted residues and further enhancing
the product by using a phage display library. Recombinant clones can be detected by
utilizing green fluorescent protein to screen colonies with ultraviolet light (Paschke et al.,
2001) or by the clever use of the chloramphenicol acetyl transferase (CAT) gene (Zahra
et al., 1999). These and others have offered new ways to deal with the problem of
19
obtaining only recombinant clones for rFab libraries that contain both heavy and light chains (Marks et al., 1991 & 1992).
Phage-displayed antibody fragments have also generated new approaches to panning as well. Three different panning methods will be described below.
An innovation in panning introduced the idea of selectively-infective phage (SIP)
(Malmborg et al., 1997, Krebber et al., 1997 &-Spada et al., 1998). A two-part control system is induced by (a) creating a non-infective phage particle by replacing the N- terminus of the gene III product with a ligand-binding protein that selectively interacts with the disabled phage particle, and (b) coupling it with an adapter molecule linked to the N-terminal region of the gene III protein thus restoring its infectivity. Only properly associated molecules will successfully infect Escherichia coli cells.
Another method is called delayed infectivity panning (DIP) (Benhar et al., 2000).
It purports to combine phage display and cell surface display of polypeptides. This system utilizes the Lpp-Omp’ hybrid fusion to produce many copies of the antigen on the surface of the F+ Escherichia coli cells. These displayed antigens attract the phage particles displaying the specific antibody to the Escherichia coli cells by specific antigen- antibody interactions thereby capturing the phages. Once captured the phages infect the cells and the cycle of enrichment for the antibody fragment of choice can be perpetuated.
Infectivity can be delayed because expression of the F pilus is temperature sensitive. At
16o C the cells are functionally F-. The phages will be bound to the cells but will not infect them. The unbound phages can be washed off. At 37o C, the F pilus will be expressed and the bound phages can then infect the cell. This method highly enriches
20
display of relevant antibody over irrelevant antibody over a million-fold according to the authors.
Finally, a third approach to improving the yields of antibody fragments selected by panning involves expressing the antigen of interest on the gene VIII product, which is the major coat protein on the filamentous phage, rather than on the few copy gene III protein (Sidhu et al., 2000). This increases avidity for the antigen. Thus a weaker binding antibody can interact with the antigen and still produce a noticeable result.
In general, for rapid and selective purification of recombinant peptide and protein products a 6-histidyl (6-His) tag is frequently added to the C-terminus and this is also true
for recombinant antibody fragments. This helps separate the recombinant protein from
other protein components on a metal chelate column in the extraction mixture. But
histidines are not neutral additions to the surface of any protein. They can be highly
positively charged depending on the buffer state of the solution in which the peptide or protein is solvated. Crystallization is frequently used as a tool to help determine the three-dimensional structure of such molecules. The surface characteristics of a protein significantly influence its crystallization properties. The shear bulk of six histidines residues may prevent favorable contacts from being made between neighboring molecules. It would be preferable if a post-translational modifier existed to selectively cleave the 6-His tag after the molecule has been purified to homogeneity. If the molecule of interest can be purified to greater than 99% homogeneity by some means other than 6-
His tagging that is preferable.
If E. coli is the source for production of the antibody fragment, this organism has a high-His protein that can be problematic as well. It is a 14 KDa protein that binds to
21
chelate columns as well as any other His-tagged protein. Its structure, function and composition are not known. Usually the 6-His tag (and not a smaller His tag) is used to help separate the molecule of interest away from the high-His protein. If a His tag as large as 6-His is not a feasible addition to the recombinant molecule, then some other means must be used to separate the alternatively His-tagged recombinant molecule from the High-His protein.
Misfolding of recombinant antibody fragments is another real problem for small libraries that are targeted to a single protein. Misfolding can lead to toxic effects on E. coli cells as evidenced by impaired or depressed growth rates in culture. In the creation of a phage-displayed antibody fragment library, misfolding leads to a functional decrease in library size. Some advances have been made in overcoming this problem. Primarily acid residues in key locations in the domain structure have been identified as the culprits in the misfolding of recombinant antibody fragments (Knappik et al., 2000 and references contained therein.). With this recently gained knowledge, better planning through bioengineering can overcome this frequently encountered problem for “one pot” libraries.
It is primarily glycosylation of the antibody fragments that may or may not delay the switch to recombinant antibody fragments in biomedical research. Glycosylation can increase the half-life of a protein or peptide significantly in the blood stream of a patient, provided that the sugar moieties are of the human type. Improper glycosylation, however, can produce adverse effects in human recipients, including shock and death reactions. Therefore, most biotechnology companies are unwilling to consider recombinant antibody fragments from species other than human or humanized cell lines
(Knappik et al., 2000 & Wang et al., 2000). However when a non-glycosylated rFab, Fv
22
or scFv is demonstrated in pre-clinical trials to be equally as efficacious as the human (or
humanized) glycosylated Fabs, then the cheaper production of this product becomes attractive.
2.3 Applications of Antibody Fragment Biotechnology
2.3.1 Biological reagents. There are many mechanisms to explain the production
of free radicals. Some products of free radicals include modifications of bases in DNA,
one of which is the oxidation of quinine to 7,8-dihydro-8-oxoguanine. Bespalov et al.,
1999, have developed Fabs specific for 7,8-dihydro-8-oxoguanine to help identify
segments of DNA that have been damaged by free radical formation. DNA damage has
been linked to cancer and autoimmune diseases. These Fabs are in the development stage
of biological reagents.
Approaches to enzyme-linked immunosorbent assays (ELISA) are notably being
revised through the use of recombinant antibody fragment biotechnology. One of the
newer developments in ELISAs is a ‘double antibody sandwich enzyme-linked
immunosorbent assay’ that utilizes scFvs for coating and detecting beet necrotic yellow
vein virus (Kersschbaumer et al., 1997). They employed the leucine zipper motif in their
design of the coating scFv to cause association between the fragments when they
interacted with their target. In general, it follows the same overall pattern for a standard
ELISA as demonstrated in Figure 2.1 (a), but the coating and detecting reagents are fully
synthetic antibody fragments. The open sandwich bioluminescent immunoassay (OS-
BLIA) is an innovative idea that eliminates all of the incubation steps associated with
ELISAs (Suzuki et al., 1999, Arai et al., 2001). For a comparison between ELISA
23
(Engvall et al., 1971, Engvall & Perlman, 1971) and open sandwich bioluminescent immunoassay (OS-BLIA) procedures see Figure 2.1.
Aa. ELISA Bb. OS-BLIA Step 1. Coat the plate with antibody (Ab). Incubate and wash away unbound Ab.
specific Antigen-specific Antigen Antigen-specific antibody Light Chain Ab Heavy Chain Ab (Ab) fragment tagged fragment tagged with enhanced with enhanced Step 2. Coat the plate with antigen, incubate yellow Renilla and wash away unbound antigen. fluorescent luciferase protein (EYFP)
antigen
Step 3. Coat the plate with enzyme-linked Ab, incubate and wash away unbound Ab.
alkaline phosphatase One Step. Mix all reagents and the test antigen. linked antibody Monitor bioluminescence resonance energy Step 4. Add p-nitrophenyl phosphate and wait for color development. transfer (BRET) within minutes.
Figure 2.1: Multi-Step ELISA Procedure versus a Single-Step OS-BLIA
Procedure. Panel A demonstrates the many steps required to complete an enzyme-
linked immunosorbent assay. Panel B illustrates the single step “open-sandwich-
bioluminescent immuno assay.”
The primary drawback of standard ELISAs is the long dwell time due to numerous cycles of binding and washing steps. As shown in Figure 2.1 panel A, ELISA
24
is a multi-step process involving numerous wash steps between each addition of reagents.
In a standard ELISA, specific antibody is bound to a well (incubated overnight at 4oC)
and unbound antibody is washed off. This is followed by a 2-hour blocking step with
BSA or milk proteins, performed at room temperature to prevent nonspecific binding to
the well, followed by its wash step. The ELISA plate is then loaded with the antigen,
incubated for 1-2 hours, followed by several wash steps. Either an alkaline-phosphatase
(Ab-AP) or horse radish peroxidase coupled antibody (Ab-HRPO), derived from an
alternate species’ monoclonal antibodies to mouse immunoglobulins, containing
specificity for a different epitope to the same antigen is then added to the plate. Finally,
the p-nitro phenyl phosphate reagent is added and its reaction product is measured at a
wavelength specific to the product of the reaction within minutes or sometimes hours
Bioluminescence resonance energy transfer (BRET) is a naturally occurring
phenomenon that does not require the use of excitation illumination. The open sandwich
bioluminescent immunoassay (OS-BLIA) is a novel approach to immunosorbent assays
that reduces the number of binding steps as shown in Figure 2.1 panel B. This method
exploits the re-association of the generally weak antibody variable region VH-VL complex by a bridging antigen. Arai et al., 2001, separately cloned the heavy and light chains of an Fv antibody fragment to hen egg white lysozyme into two separate E. coli Origami
(DE3) cell lines. They modified its Fv light chain to contain an enhanced yellow fluorescent protein (EYFP) on the N-terminus (VL-EYFP). They also modified the Fv
heavy chain to contain Renilla luciferase on its N-terminus (VH-luc). The VL-EYFP is
mixed in solution with the lysozyme, the VH-luc and coelenterazine (luciferase substrate).
When the VH heavy and VL light chains associate to form the Fv in the presence of the
25
hen egg white lysozyme antigen, bioluminescence resonance energy transfer occurs between the EYFP and the Renilla luciferase that can be measured (Suzuki et al., 1999,
Arai et al., 2001).
2.3.2 Therapeutics and diagnostics. Therapeutic antiviral applications are
sought because they are the least tractable to antibiotic therapy. The primary areas of
antiviral research are viral caused cancer, AIDS and hepatitis B and hepatitis C. Cancer
has many causes, but if a specific virus is associated with a cancer (like Rous sarcoma
virus), then anti-viral therapeutics are appropriate. There are other viruses, such as
Ebola, that cause human and animal disease. In the area of cancer therapeutics some novel ideas are being explored, such as the use of immunotoxins (immunoglobulin
fragments fused to toxins or radioisotopes, as illustrated in Figure 1.4 (d)) to produce a
tumor-targeted response in some types of cancers (Jung et al., 1999, Kaminski et al.,
1993, Martineau & Betton, 1999, Pai et al., 1996, Poul et al., 2000 & Press et al., 1993).
When the fusion product does not immediately produce something useful,
computer design, genetic engineering and recombinant protein expression can improve
on the product as demonstrated by Chowdhury et al., 1998 and Arkin & Wells, 1998.
Stability engineering is resorted to when a good idea for a therapeutic antibody fragment
does not yield a stable product in a prokaryotic cell line. Acid residues seem to be the
most vulnerable. Stability engineering in scFvi antibody fragments seems to be a major
area of focus in the biotechnology industry. See the article by Wörn & Plückthun, 2001,
and references contained therein.
Fully synthetic human combinatorial antibody libraries (HuCAL) are also being
developed for expression in Escherichia coli. Knappik et al. have made a thorough study
26
of the most common sequences utilized in the human repertoire of immunoglobulins.
They included all available immunoglobulin types in their study and based their final
choices on modular consensus frameworks and the randomization of the complementarity
determining regions with trinucleotides. The most common human motifs in the design
of these antibody fragments may help minimize possible “non-self” reactions to their products similar to what one would see in a mismatched blood transfusion or organ transplant. More simply the authors probably wanted to have an economical number of antibody fragments as a usable subset of all the possibilities. These HuCALs are being developed for their potential use in cancer therapeutics.
Diagnostic applications constitute a major area of research in the biomedical industry. HER2 (human epithelial growth factor receptor 2) is frequently over-expressed in various cancers, but it has been studied most in conjunction with human breast cancer. The anti-HER2 mFab is being utilized for diagnostic purposes to detect increased levels of
HER2 expressed on the surface of breast tumor cells. The tissue section is visually scored from 0 to 3+ by immunohistochemical detection of HER2. Bera et al., 1998, are looking at
anti-erbB2 as a tumor therapeutic because erbB2 is elevated in some cancer patients.
Vascular endothelial growth factor (VEGF) is increased around tumors that stimulate blood
vessel growth around them thereby enhancing their supply of nutrients. Thus another
tumor-specific antibody fragment being developed is anti-VEGF (Chen et al., 1996.)
Breast tumors are also therapeutic targets (Dokurno et al. 1998).
Therapeutic applications of antibody fragments are being sought for many disease
states including, but not limited to, cancer, hepatitis, arthritis and multiple sclerosis.
Currently there are two humanized monoclonal antibody fragments specifically designed
27
for cancer treatment, anti-CD20 (RituxanTM) and anti-HER2 (HerceptinTM) (also used as a
diagnostic reagent above). See the following web sites: http://www.herceptin.com and http://www.gene.com/gene/products. An scFv is generally thought to be more able to
penetrate a tumor because of its smaller size than an Fab. Converting an Mab to an scFv
and fusing a toxin to it is a means of directing a highly specific therapeutic to a tumor
(Chowdhury et al., 1998). Direct intervention in the metabolic pathways associated with
cancer is also being researched with the aid of phage-displayed Fab antibodies (Horn et al.,
1999).
Patients suffering from rheumatoid arthritis, multiple sclerosis or vasculitis are
benefiting from a product developed primarily against lymphoma (Press et al., 1993).
The humanized form of the rat anti-CD52 (CAMPATH-1) attaches itself to the smallest
known cell-surface glycoprotein antigen CD52. CAMPATH-1’s presence on the surface
of the lymphocyte kills the cell by either a complement-mediated or cell-mediated lysis.
It is also used in the treatment of marrow and organ transplantation (Cheetham et al.,
1998, and references contained therein).
Hepatitis C is a serious disease worldwide and gaining ground in the United
States as well. One drug target is inhibition of the hepatitis C virus (HCV) NS3 protease.
The Fv fragment of antibody 8D4 was derived first from immunization of animals then
converted to an scFv. The Fv fragment was not very soluble, but engineering it into an
scFv improved its solubility but decreased its affinity for its target activity (Kasai, et al.
2001). Another target is NS3 helicase to which five scFvs cloned from humans with the
disease are also under investigation as possible therapeutic tools (Tessman et al., 2002).
28
Novartis is marketing SimulectTM, a recombinant chimeric (mouse/human)
monoclonal antibody that binds to and blocks the interleukin-2 receptor α-chain on the
surface of activated T cells for use in management of acute organ rejection in transplant
patients. The production of human anti-mouse antibodies (HAMA) may cause a
hypersensitivity reaction in some patients and is one of the primary drawbacks to
developing humanized mouse recombinant antibody fragments. (See the following web
site for more details: http://www.pharma.us.novartis.com/product/pi/pdf/simulect.pdf ).
2.3.3 Catalytic recombinant Fv antibody fragments. Catalytic antibodies were discovered in the 1980’s and by 1991 recombinant catalytic antibody fragments were being designed via protein engineering as well as by ligand or hapten-based approaches
(Jacobs, 1991). Analysis of hapten binding and catalytic determinants has been carried out in a family of catalytic antibodies called oxy-Cope antibodies designed for studying the immunological evolution and sequence diversity of this series (Ulrich & Schultz,
1998). A catalytic Fv fragment intrabody (estereolytic antibody) has been developed recently (Ohage et al., 1999) and the catalytic 43C9 Fab antibody has been utilized to determine the structural basis for amide hydrolysis (Thayer et al., 1999).
2.3.4 Epitope mapping of difficult-to-crystallize proteins. Glycoproteins are often refractory to crystallization or when they do crystallize, diffract too poorly to obtain the three-dimensional structure. Many are too large to apply 2-D or 3-D NMR techniques. Epitope mapping is an alternative method compared with NMR or x-ray crystallography. A 120 KDa glycoprotein (gp120) from HIV has been mapped utilizing scFvs from a phage-displayed library (Ditzel et al., 1997). The major drawback of epitope-mapping is the limited amount of structural information it yields. This is a shot
29
gun approach to structure because one is aiming the entire immune system at one target
molecule’s antigenic determinants hoping to define the surface of that molecule indirectly
through the polyclonal antibodies formed against it. This is evidence that we need better
ways of crystallizing glycosylated proteins.
2.3.5 Structure enhancement or structure determination. Whether planned or
accidental, mutants of recombinant antibody fragments can lead to increased thermo-
dynamic stability with improved folding. An scFv mutant of human anti-β−galactosidase
demonstrated an equal rate of expression in Escherichia coli cytoplasm to the native form
of the scFv, but it folded correctly in the cytoplasm and the unmutated scFv did not
(Martineau & Betton, 1999).
There are at least four crystal structures of one scFv and three phage library-
derived Fv antibody fragments complexed with turkey or chicken egg-white lysozyme,
(Ay et al., 2000, Bhat et al. 1994, Cohen et al., 1996 & Padlan et al., 1989). Primarily
these fragments have been utilized to try to determine how antibodies recognize an
epitope. Additionally, Ay et alii demonstrated that the level of specificity could be
reinforced by the addition of the light chain variable domain.
2.3.6 Research tools. Recombinant antibody fragments have been developed as
research tools for studying amide hydrogen exchange rates (Williams et al., 1997), for
kinetic analysis (de Haard et al., 1999 & Scheuermann et al., 2003) and for understanding
disease processes in general. Hapten structure determinations of peptide hormones
(Ulrich & Schultz, 1998), for example, and genetic diversity engineering of one-pot
libraries specifically to meet a research need are also common applications of this new technology (de Haard et al., 1999). Anti-idiotypic antibodies are being used as “tools for
30
a better understanding of molecular mimicry and the immunological network” (Arkin &
Wells, 1998, Goletz et al., 2002) and also are being investigated as therapeutic tools in
autoimmune disease (Escher et al., 2002).
Phage-displayed Fabs or scFvs are being developed for use as probes for basic research in structure and/or function. One such scFv library is probing turkey egg-white lysozyme (Ay et al., 2000). Another is probing second sphere residues in an anti- estereolytic antibody (Arkin & Wells, 1998). Other such probes have been developed
against human dendritic cell surface antigens (Lekkerkerker & Logtenberg, 1999).
It is not enough to have the sequence of a genome. Effective research tools are being developed to study the function and cellular location of the gene products.
Innovators have devised a method utilizing scFvs to study gene expression (Cyr &
Hudspeth, 2000, Krebber et al., 1997, Siegel et al., 2000 & Walter et al., 2001). Cyr &
Hudspeth are probing the rare inner ear proteins utilizing their scFvs designed specifically for that purpose using a multi-step process involving animal immunization.
It is quite tedious to have to immunize a different species of animal each time a new research tool is developed. Phage display of multiple scFvs and panning to improve the yield of high-binding scFvs are attractive tools because the possibility of the elimination of intermediate animals and the speed with which they can be generated. Phage display libraries in the range of 105 to 1010 are practically obtained in a typical scFv library.
Fusing a specific scFv to a reporter protein like green fluorescent protein or a
radionuclide is a highly sensitive method of localization of the antigen within a cell.
Phage display is not the only display system. Antibody fragments with capacity to specifically bind Staphylococcus aureus protein A-based affibodies have been
31
displayed on the surface of staphylococci in an effort to create whole-cell diagnostic devices (Wernérus et al., 2002, Gräslund et al., 2002).
2.3.7 Affinity enhancement. Some antibody fragments have a higher affinity for their cognate antigen than others. When the antigen is a hapten like estradiol it has very little chemical difference from other estrogens or even testosterone. By randomizing insertions into the second heavy chain CDR loop Lamminmaki and coworkers have enhanced the affinity of anti-estradiol antibodies so that they no longer recognize
testosterone (Lamminmaki et al., 1999).
2.3.8 Control of plant pathogens. Plant pathogens can destroy acres of crops if not prevented. Plants themselves are being genetically designed to contain antibodies against naturally occurring plant pathogens producing “plantibodies” (Schots et al.,
1992). The mode of transformation is by “transfecting plants with genes encoding
monoclonal antibodies against pathogen specific proteins.” Recently an scFv antibody
fragment has been constructed against a plum pox viral RNA replicase Nib. Schots et al.,
1992, are employing an immunomodulation approach for engineering plant virus resistance. Transgenic plants expressing recombinant antibodies however may only be
partially protected against infection (Esteban et al., 2003, Fecker et al., 1997, Schillberg
et al., 2000, Tavladoraki et al., 1993, Voss et al., 1995 & Zimmerman et al., 1998).
Esteban et al. point out that targeting the viral replicating enzyme system should be a
more effective way to interfere with viral infection.
2.3.9 Biosensors. Another hot topic in today’s research is the development of
antibody fragments as biosensors. The main considerations are sensitivity, selectivity,
cost and stability of the cloned fragments (Hock et al., 2002 & Xiao et al.,2000). Speed
32
of detection is a variable that has not yet been addressed. They are apparently designing antibody fragments as components of sensor chips (Hock et al., 2002), but currently one does not have any instrument available for immediate detection of toxins or other bioterrorism agents in a field situation.
2.4 Future Applications and Developments in Biotechnology
There are numerous commercial reagents in the laboratory catalogs for monoclonal antibody fragments. It is just a matter of time before their recombinant equivalents will become available. For example, the OS-BLIA (Suzuki et al., 1999) holds great potential for replacing ELISAs in the not too distant future.
As the article by Kerschbaumer et al., 1997, implies, there is a broader range of use for recombinant antibody fragment biotechnology than just human disease.
Agricultural interests in crop plant pests and domestic animal pests and their respective diseases also require diagnostic and therapeutic applications to be developed. Many of the applications developed by biotechnology companies for the farming industry could very well include antibody fragments and fusion proteins such as immunotoxins.
Alternatively, plants are an even cheaper source of protein production. Currently, potatoes and corn are being studied as sources for antigen production to create safe vaccines for immunizing the world’s population against diseases. If these vaccines are found to be safe and efficacious in humans, then that opens up the possibility of new sources for recombinant antibody fragments as well.
Xenotransplantation research is directed toward the hope that someday the deficiency in available organs for transplantation in humans can be supplied by another
33
species. Pigskin has been used as a temporary covering for burn victims and pig hearts
are the closest to human in their overall size, weight and vascularization. Studies on
rabbit anti-guinea pig C3 suggest to this author a method for monitoring complement C3
involvement in organ rejection (Hawlisch et al., 2000). Human anti-pig C3 could be
monitored instead. This reagent would have to be coupled to something like the OS-
BLIA reagent described earlier for a timely diagnosis to be made. In xenograft rejection
one does not have days to take remedial action. Perhaps prophylactic human anti-pig
heart scFvs fused to their human antigen counterparts could be developed to coat the pig
antigens with human antigens in order to disguise the pig heart as a human heart and thus
prevent rejection.
Only transplanted organs between identical twins are truly identical in every
genetic detail. Most organ transplants are from nearest serotype donors. Even the best
cross match between non-related donors and recipients cannot eliminate the need for
immune suppression of the organ recipient. The use of humanized Fabs or scFvs of any
or all antibody types (Boel et al., 2000) may be adapted to prevent allograft rejection.
Once developed and proven safe, their addition to the prophylactic chemotherapeutic
regimen that organ allograft transplant recipients receive could reduce the necessity for
stronger chemotherapeutic medications that have such severe side effects. Other
unforeseen human therapeutic uses may also develop along those lines.
Carcinogenic tumors need a lot of nutrients to support their growth. Usually these
tumors have more than their share of blood vessels to supply these nutrients. Diagnostics
for carcinogenic tumors by in situ preparations of the tissue may be enhanced by the use
of phage-derived antibodies specific for angiogenic or other specific markers coupled
34
with infrared photo detection methods (Pai et al., 1996, Birchler et al., 1999). This could
also lead to a tumor-specific therapeutic delivery system for targeting fast growing
tumors. Recombinant anti-idiotypic antibodies have been found that inhibit a certain
class of auto antibodies which may help develop a therapeutic strategy for chronic
immune thrombocytopenic purpura (Escher et al., 2000).
Some phage display libraries being developed now have great potential for
agricultural, veterinary and biomedical applications. The sheep scFvs being generated
are a system of monoclonal derived antibodies that are simpler to work with than either
mouse or human antibodies because of their lesser number of diversity elements (Li et al., 2000).
Another area not often discussed is the need for detection of biological toxins in water and food supplies. Research in this area may lead to very sensitive biosensor devices if the antibody fragments can be coupled with a portable detector system
(McElhiney et al., 2000). Work is ongoing in the area of biosensor development because bioterrorism has become a reality in the recent news. People who never heard of anthrax prior to a few years ago now can identify what this organism is. Also, our current U. S. government administration keeps emphasizing bioterrorism as a world threat. Biosensor development hinges on utilizing antibody fragments because of their sensitivity and specificity for their respective targets (Siegel et al., 2000). One of the factors in developing biosensors is fabrication cost for sensor chips. Another factor is sensor stability. Both of these factors are being addressed by Kramer et al.,2002, by switching from a less stable scFv and more expensive production system to a more stable Fab
35
fragment in a cheaper induction system that utilizes a bioreactor for production (Walter et
al., 2001).
Much progress has been made through the availability of x-ray crystal structures
of proteins. Thanks to progress in computer technology and programming, the rate-
limiting step is now the crystallization of the protein of interest when adequate supplies
of the protein exist. Only about 10-20% of all proteins actually crystallize utilizing the
standard array of available crystallizing reagents. Better reagents to crystallize proteins
(and other biological polymers) are needed for those that fail to crystallize. Recombinant
Fabs with their high affinity for specific molecules are a natural choice for this
application (Kovari et al., 1995). The problem is to find one with ideal crystallization
properties that are reproducible regardless of the antigen so that a high-throughput
crystallization technology can be developed.
No discussion of future directions in biotechnology would be complete without
mention of the companies that are involved in these efforts. New companies have been
formed, patenting numerous aspects of recombinant antibody fragment biotechnology.
Patenting these ideas could be very lucrative for some of these new biotechnology
companies, but they could also fail for lack of good management and the timely bringing
of new products to market.
Germany is a hotbed of biotechnology hopefuls. One such company is called
Diversys (http://www.diversys.co.uk/applications.htm). They mention their patents and
that they produce recombinant Fabs and scFvs but their main product emphasis seems to
be on the product SuperantibodiesTM, claiming that they are recombinant antibodies with built-in expression, folding and superantigen binding characteristics. They expect to be
36
able to make recombinant antibodies to any existing antigen and to market them.
Another young company founded in 1998 in Berlin, Germany is GenPat77 to treat
immune disorders (http://www.genpat77.de/home/index.htm). Connex was founded in
1994 as a diagnostics and bio-pharmaceuticals company based on antibodies and their
derivatives (http://www.connex.de). One of the very newest biotechnology companies is
NEMOD GmbH, founded in January, 2000. Their focus is on the development of new
and innovative diagnostics and therapeutics with special emphasis given to clinically
relevant cancer-associated antigens with special emphasis on carbohydrate antigens
(http://www.nemod.de/index.html).
2.5 Summary
Recombinant antibody fragments (rFabs) have a much greater potential than
monoclonal antibody fragments as aids to structure determination, as laboratory tools,
and for diagnostic and therapeutic applications because of their ease of production and
the greater degree of control the bioengineer can exercise over the final product. Many of
the problems associated with recombinant antibody fragment biotechnology have already
been overcome. And, there are many current applications of antibody fragment
biotechnology including biological reagents, therapeutics and diagnostics, catalytic
recombinant Fv antibody fragments, epitope mapping of difficult-to-crystallize proteins,
structure enhancement or structure determination, as research tools and for affinity
enhancement. Antibody fragments as biosensors are just coming on the scene and their
development is a high priority for anti-bioterrorism efforts ongoing within the military.
37
CHAPTER 3
OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION OF
MOUSE RECOMBINANT ANTIBODY FRAGMENTS IN ESCHERICHIA COLI
3.1 Overview
In this chapter the methods of obtaining a usable mutant for the basis of a co- crystallization library will be presented along with the corresponding results. Overall, four mutants were constructed (rFab2-rFab5) from a mouse recombinant antibody fragment (Mab25.3) whose epitope was HIV I p24 capsid protein. Three mutants (rFab3- rFab5) were constructed and optimized by the author of this dissertation for secretion of rFab to the periplasmic space of Escherichia coli, but mutants rFab2, rFab3, rFab4 and rFab5 also expressed rFab to the medium. The mutants numbered 2 and higher were His- tagged to aid in purification. These four mutants were developed and tested (rFab2 through rFab5) for stability, ease of production, yield and crystallizability. We discovered that Escherichia coli strains BL21 (DE3) and BL21 (DE3)-RIL exported the rFabs to the medium. Whereas rFabs were detected in both super broth and defined media by Enzyme-linked ImmunoSorbent Assays (ELISAs), defined medium provided a superior medium for isolation of the rFabs. A rapid purification protocol was developed for extraction of pET28 derived-Fabs from the medium. Confirmation of the identities of
38
the rFabs was by DNA PCR and protein SDS-PAGE. Correctly folded antibody
fragments were detected by an indirect ELISA with the HIV I p24 capsid protein antigen forming the first layer of the sandwich.
Crystallizability of the uncomplexed rFab mutants was the main deciding factor in the selection of the specific rFab mutant for use in building a co-crystallization library. rFab4 was selected over the other mutants primarily because of its relatively high yield and its ability to form small crystals. The other mutants either did not express well or crystallized at such a slow rate as to make them unfeasible as co-crystallization reagents.
3.2 Rationale
Previously, a monoclonal mouse antibody was papain digested, purified and co- crystallized with its cognate antigen, HIV-1 p24 capsid protein (Momany, 1996). This mouse monoclonal Fab was derived from an IgG. It has a κ light chain and a truncated γ heavy chain for a nominal molecular weight of 50 KDa. The mFab-p24 structures are available from the Protein Data Bank (PDB) under identifier number 1AFV at http://www.rcsb.org/pdb/cgi/queryForm.cgi (Abola et al., 1987) and form the basis of the
recombinant antibody fragment that is the topic of this dissertation (Momany et al.,
1996). Currently 37 other monoclonal Fab crystal structures are also available in the
PDB for comparative studies, if needed.
Our goal was to create a recombinant antibody fragment that was easy to purify in
large quantities and that would crystallize readily in the same space group with the same
cell constants as its parent monoclonal antibody fragment. This would make structure
determination by molecular replacement relatively simple. Recombinant DNA can be
39
easily manipulated via PCR to produce mutants that are frequently more useful than the
original sequence.
Four mutants were created. Recombinant Fab2, created by Dr. Momany, had a 6-
His tag on the heavy chain to aid in purification of it. Recombinant Fab3 had 2-His tags on both chains at the C-termini to create a vicinal His-tag that was less bulky but still
effective as a purification handle. Fab4 had a correction mutation in the heavy chain and
Fab5 had a heavy chain mutation (N57K) to induce the rFab molecules to self-assemble
in crystals and a silent mutation adjacent to it to make its clone’s DNA distinguishable
from Fab4 DNA by restriction endonuclease fingerprinting.
3.3 Materials and Methods
3.3.1 Chemicals and biological reagents. Kanamycin, chloramphenicol,
isopropyl-β,D-thiogalactopyranoside (IPTG), ethylenediaminetetraacetic acid (EDTA),
ammonium sulfate, phenylmethylsulfonyl fluoride (PMSF), yeast extract and tryptone were purchased from Fisher Scientific. Vitamins and L-amino acids were a gift from
Michelle Momany. Sepharose 6B gel and divinyl sulfone were purchased from Sigma-
Aldrich. Buffer and reagent chemicals were of the highest analytical or molecular grade available from commercial vendors.
The original mouse monoclonal antibody cell line containing its specific kappa light chain and gamma heavy chain for Fab25.3 was a gift from Jan McClure. The pCOMB3H phagemid was received under a limited use agreement from Dr. Carlos
Barbas, III, at Scripps Research Institute in La Jolla, CA. Both the pET28b vector used for cloning and the host bacteria (E. coli XL1-Blue) used for propagating the dsDNA
40
pET28-Fabs were from Stratagene (La Jolla, CA). Expression of antibody fragments was carried out in the low-protease strains of E. coli BL21(DE3) and in E. coli Codon Plus
BL21(DE3)-RIL (with rare Arg, Iso and Leu mRNA codons) (Stratagene, La Jolla, CA).
For simplicity, the host strains of E. coli BL21(DE3) will be referred to as DE3 and E. coli Codon Plus BL21 (DE3)-RIL will be referred to as RIL. Plasmid Mini and Midi kits and QuikChange Site-Directed Mutagenesis kits were obtained from QIAGEN, Inc., and used according to the manufacturer’s directions for DNA preparation and mutagenesis.
Protein molecular weight markers were purchased from Pharmacia.
Deoxynucleotide (dNTPs) mixes were purchased from Stratagene or Boehringer.
Restriction endonucleases, Xba I, Sac I, Xho I, NheI, Van 91 I and Spe I, were purchased from Boehringer. Oligonucleotide primers were synthesized on a DNA synthesizer at the
Molecular Genetics Instrumentation Facilities (MGIF), University of Georgia, Athens,
GA. The QuickChange Site Directed Mutagenesis Kit was used to produce the various pET28-rFab mutants. The chemically competent E. coli XL1-blue cells that came with the QuickChange Site Directed Mutagenesis Kit were used for the transformation of the double mutant pET28-Fab3 following their protocol. Other transformations were done using electrocompetent cells.
Oligonucleotide production and purification and cDNA clone sequencing were services provided by MGIF on campus. Low molecular weight and high molecular weight PCR standards were purchased from Promega. Agarose gels were poured with
TBE buffer and ethidium bromide to dye the DNA bands and the bands were visualized with a Kodak camera. (see Appendix B for more details).
41
3.3.2 Preparation of rFab expression plasmids for optimized protein
expression and crystallization. Recombinant Fab plasmids were generated by
polymerase chain reaction (PCR)-based mutagenesis using the QuickChange™ kits by
Stratagene. The PCR-based mutagenesis was utilized to introduce single or multi-site
mutations to the pET28 vector. PCR amplifications were performed with a RoboCycler
Gradient 40TM (Stratagene). Four mutants were designed, created and tested for their
suitability for making an Fab antibody fragment library. Plasmids were transformed by
electroporation into E. coli XL1-blue for cloning or into BL21 (DE3) or BL21 (DE3)-RIL strains for high-level protein expression. (See Appendix B Short Protocols for details of electroporation transformation of E. coli.)
The initial Fab from which this work is derived was constructed by Dr. Cory
Momany including the transfer of the cloned light and heavy chain sequence from the pCOM-Fab2 into the pET28 plasmid (using the techniques reviewed by Capechi, 1989 and Koshland, 1989). Subsequent modifications were performed by the author of this dissertation on the pET28 plasmid mutant pET28-Fabn, where n refers to the sequential number of the modified mutant 3 through 5. Primers M2H2DH-F and M2H2DH-R were designed to mutagenize the heavy chain, and primers M2L2DH-F and M2H2LH-R were used to mutagenize the light chain. Two PCR reactions with a QuickChange™ kit produced the double mutant Fab3. Primers HCREVDELL and HCFORDELL were used to create the Fab4 deletion mutant. FAB5FRWD and FAB5REV primers were used to accomplish the construction of the Fab5 mutant. See Table 3.1 for primer sequences.
Single colonies were picked from freshly isolated, transformed XL1-blue, DE3 or
RIL cell lines, cultured in LB broth overnight at 37oC on a shaker with appropriate
42
Table 3.1: Primer Sequences for PCR Mutations to pET28 Plasmids.
pET28-Fab3
M2H2DH-F 5’CCA-GGC-CTT-ACC-AGC-ACA-GAC-CCA-GGC-TGC-
TCG-AGC-TGC-ACC-TGA-TCG-A-3’
M2H2DH-R 5’CCC-CAT-CAT-ACT-AGT-TAA-GCG-GCC-GCA-GCT-
AGC-3’
M2L2DH-F 5’-CTC-CAC-GTC-GAC-GAG-CTC-GTC-GGA-CCC-AG-3’
M2L2DH-R 5’-CAT-GAA-TCA-CCA-CTA-ATC-TAG-ATA-ACC-ATG-
GG-3’
pET28-Fab4
HCREVDELL 5’GAC-CCA-GGC-TGC-TCG-AGC-TGC-ACC-TCG-GCC-
ATG-3’
HCFORDELL 5’CAT-GGC-CGA-GGT-GCA-GCT-CGA-GCA-GCC-
TGG-GTC-3’
pET28-Fab5
FAB5FRWD 5’GTT-AGT-ATT-ACC-ACT-CTT-TGG-ATG-AAT-CTC-
TC-3’
FAB5REV 5’GAG-AGA-TTC-ATC-CAA-AGA-GTG-GTA-ATA-CTA-
AC-3’
LC is light chain; HC is heavy chain. Restriction sites are underlined.
43
antibiotics and diluted by half with a sterile solution of 65% glycerol in 20 mM tris·HCl,
pH 8.0. The E. coli cultures were stored in the –80oC freezer for long-term storage. Agar
plate cultures were maintained for intermediate storage of a few weeks in the 4oC cold
box.
3.3.3 Production of rFab proteins. A schematic of the process of protein induction and purification is shown in Figure 3.1. Protein production of the various rFabs
was carried out in the following manner.
Super broth (SB) for culturing E. coli consisted of (30 g of tryptone, 20 g yeast
extract and 10 g of MOPS) per liter and titrated to pH 7.0. Defined medium contained
(per liter) 4 g glucose, 2 g NH4Cl, 6 g KH2PO4, 13.6 g Na2HPO4 anhydrous or 25.6 g
Na2HPO4 heptahydrate, each of the amino acids at 4 mg/L and four vitamins at a
concentration of 1 mg/L, plus 0.02 M MgSO4 and 25 mg/L FeSO4 x 7H2O. See
Appendix B for details of preparation.
Twenty micro liters (20 µl) of an inoculum were directly transferred to 100 ml of defined medium and incubated for 12 to 18 hours at 37oC before transfer to a 2.8 L
Fernbach flask containing 1 L of fresh defined medium. The optical density was
measured at 600 nm at various intervals using an Ultraspec 2000 UV/Visible
spectrophotometer (Pharmacia Biotech) to follow cell growth. After two hours of growth
at 37oC the cultures were rapidly cooled in an ice bath and grown for one hour at room temperature. Protein production was induced with 0.5 ml of 1.0 M IPTG (Fisher
Scientific). Induction was stopped at the predetermined intervals, usually 6 hours later
but could go as long as 24 hours. 0.5 ml of 10 mM PMSF dissolved in isopropanol was
44
added to inhibit protease activity in the medium. The cultures were centrifuged at 6000 x
G for 30 minutes at 4oC to separate the cells from the medium.
There were two sources from which to extract rFab protein, the E. coli periplasm and the medium in which it was grown. The following precipitation, dialyses and centrifugation steps were maintained at 4oC. Periplasm extraction of rFab mutants from resuspended cells was performed over ice for 30 minutes in 10 milliliters of periplasm extraction buffer (0.2 mg/ml lysozyme, 1 mM EDTA, 0.5 µM PMSF, 1.0 M NaCl, 50 mM tris HCl, pH 8.0) per gram of cells (wet cell weight). The solution was centrifuged for 30 minutes at 6000 x G for 10 minute and the pellet was discarded. The proteins were precipitated by the addition of solid ammonium sulfate to achieve 70% ammonium
E.coli culture with crFab construct 6,000 x G Separate Cells from Defined Medium
4oC Centrifuge 80% Ammonium Sulfate Precipitation
o 37 C 6-hr IPTG overnight induction 50,000 x G RT
Dialyze the precipitated HPLC proteins against HPLC 4oC Centrifuge Eluent buffer A which resolubilizes many SDS-PAGE Crystallize proteins including crFab. ELISA crFab
Figure 3.1: Protein Induction and Purification Process. See text for details of the process.
45
sulfate saturation. The precipitate was resuspended and dialyzed against the HPLC buffer.
The proteins produced in the E. coli supernatant were precipitated overnight by the addition of solid ammonium sulfate to achieve 80% ammonium sulfate saturation.
The precipitated proteins were collected by centrifugation at 6,000 x g in a JA-10 rotor.
The ammonium sulfate precipitated proteins were resuspended and dialyzed three times against an appropriate eluent buffer at pH 7.4 that resolubilized the rFabs and removed some of the undesirable E. coli proteins. The first dialysis buffer was supplemented with
5 mM EDTA in either 0.05 M Na phosphate, pH 7.4 or 5 mM EDTA in 0.02 M tris·HCl, pH 7.4,o to remove bivalent cations in the medium that would interfere with the metal- chelate column. The two subsequent changes of dialysis buffers [buffer for A11 in the high performance liquid chromatography (HPLC) purification steps to follow, 0.05 M Na phosphate, pH 7.4 or 0.02 M tris·HCl, pH 7.4] were used to remove the EDTA. A minimum of 8 hours of dialysis was needed to accomplish the removal of metal ions and
EDTA. The dialyzed sample was clarified prior to loading it on the HPLC instrument by centrifugation at 50,000 x g.
3.3.4 Protein Purification Methods. HPLC buffers for the purification process were changed as the protocol evolved. Phosphate crystallizes readily in the presence of any bivalent cation. Phosphate in the HPLC buffers had to be dialyzed out or exchanged prior to further purification on the Q sepharose column or before crystallization trials.
The obvious solution was to eliminate it from the HPLC buffers. HPLC was performed on an Äkta Purifier (Pharmacia Biotech). HITRAPTM metal chelate and HITRAPTM Q
46
ion exchange columns were used. Three methods that were utilized to purify rFabs
follow.
3.3.4.1 Two-Step Purification of rFab by NTA Column Followed by Q
Sepharose Purification. Originally the HPLC purification method involved only an
NTA metal chelate column with buffer A11 (0.02 M Na phosphate, pH 7.4) and buffer
B1 (0.5 M imidazole, 0.02 M Na phosphate, pH 7.4) forming the gradient. This purified
the proteins to about 50%-90% of homogeneity depending on the source utilized. This had to be followed by Q sepharose column purification to improve the protein purity to
95%-98% homogeneity. Between the two columns was another dialysis step to exchange
the buffers into the starting buffer for the Q column. The second purification step was
done with a Q Sepharose column. Buffer A1 for Q separation was 0.02 M tris·HCL pH
8.0 and buffer B1 was 1.0M NaCl, 0.02 M tris·HCL, pH 8.0, to separate the rFab from
the remaining E. coli proteins on the basis of charge. The flow rate for loading the
sample was usually 1.25 ml/min.
3.3.4.2 Purification of rFabs by Coupled Q-NTA Columns. This method
involved two columns, a HiTRAP Q and an NTA chelating (Pharmacia, Corp.),
piggybacked together for separation by HPLC and three buffers A11, A12 and B1.
Buffer A11 was 0.1 M NaCl, 0.02 M tris·HCl, pH 7.4; buffer B1 was 0.5 M imidazole,
0.02 M tris·HCl, pH 7.4; and A12 buffer was 1.0 M NaCl, 0.5 M imidazole, 0.02 M
tris·HCl, pH 7.4. The flow rate for loading the sample was also 1.25 ml/min. The
program fab5QNi was written to automatically switch between the A11 and A12 intake
lines. This single step alone purified the protein to 95%-98% of homogeneity.
47
To achieve a higher level of purity, a second HiTRAP Q column was employed
after dialysis into the A1 buffer using a gradient formed between bufferA1 (20 mM
tris·HCl, pH 9.0) and B1 (1.0 M NaCl in 20 mM tris·HCl, pH 9.0.). This would further
purify the rFab to 98-99% homogeneity. The same flow rate was used as that above.
3.3.4.3 Purification of rFab by T-gel. A more recent protocol considered the
use of T-gel for a rapid purification protocol to separate out rFab directly from the
defined medium with the addition of solid ammonium sulfate to the medium (25% of
saturation). T-gel was prepared under the hood with sepharose 6B gel and divinyl
sulfone following the Hutchens’ protocol (1992). T-gel Buffer 1 was 1.0 M (NH4)2SO4,
0.05 M NaxHxPO4, pH 7.5. T-gel Buffer 2 was 0.05 M NaxHxPO4, pH 7.5. It may be
noted that this was a hydrophobic interaction purification scheme. The sample load flow
rate was 10 ml/min.
3.3.5 Detection of rFabs by Native PAGE, SDS-PAGE gel analysis and
ELISA. Electrophoresis and gel staining of native and sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) gels for protein detection were
performed utilizing a Pharmacia Biotech PhastSystem TM.
The native gel required native buffer strips and a precast 12.5% gel. For the
native gel equimolar amounts of rFab3 with p24 and rFab5 with p24 were incubated
overnight in 37ºC water bath by the lab technician (Betty Ngo). The p24 solution was 30
mg/ml (1.25 mM.) The volumes of 10 mg/ml rFab3 (0.21 mM) and 7.86 mg/ml rFab5
(0.16 mM) were adjusted accordingly. The native gel did not use any reducing agent or
sodium dodecyl sulfate. After electrophoresis the native gel was silver stained. The SDS-
PAGE gels required SDS buffer strips and 20% or high density SDS polyacrylamide gels.
48
The proteins were boiled for 3 minutes together with 2x SDS bufferand electrophoresed
using the appropriate program on the PhastSystem. The SDS-PAGE gels were silver
stained.
The solid matrix for ELISA was Immulon II TM 96-well EIA/RIA plates
purchased from Fisher Scientific. ELISAs were performed using either goat anti-mouse
IgG F(ab)2 linked to alkaline phosphatase (GAM-AP) or polyclonal rabbit anti-mouse
IgG linked to alkaline phosphatase (RAM-AP) (Engvall et al., 1971) from Pierce
Chemical, p-nitrophenyl phosphate (pNPP) was from Sigma. See Appendix B for a detailed ELISA protocol specific for our inverted-sandwich type of ELISA.
3.3.6 Crystallization of rFab Mutants and Data Collection. Crystallizations were set up in a 24-well Linbro tissue culture plate using 1 ml solutions in the well and glass or plastic cover slips. A 2 µl drop of rFab protein solution was placed on the cover slip. A 2 µl drop of well solution was added to it and the cover slip was then placed atop
the well sealed with either grease or petroleum jelly. Crystal Screen I kit was used for a
series of crystallizations with rFab3. rFab4 was prepared and sent to George DeTita,
Hauptman-Woodward Institute, Buffalo, NY, for a larger 1536-well screen for
crystallization conditions using a high-throughput robot.
X-ray data were collected in Professor B. C. Wang’s x-ray laboratory on a Rigaku
RAxis4 area detector with a CuKα rotating anode that utilized graphite mirrors to focus
the x-rays. Data for the rFab4 crystal were processed using Denzo.
3.3.7 Diaminopimelate/IPTG induction of Fab3. One of the preliminary
experiments with pET28-Fab3 addressed the question of what factors would improve the
expression of rFabs. The factors evaluated included IPTG induction with or without
49
diaminopimelate, a cell-wall component, as well as the use of defined medium or super
broth as the medium of choice. There were twelve E. coli RIL cultures started from a single broth culture grown up overnight to an O. D. 600 nm of 2.0. 100 µl of this start
Table 3.2: Design of the Diaminopimelate/IPTG experiment.
Start medium to
scale-up No additives 0.5 mM DAP 0.5 mM DAP + 0.5 mM IPTG
medium 0.5 mM IPTG
10x → 1 x
vitamin defined Culture A Culture B Culture C Culture D
medium
1x → 1x
vitamin defined Culture E Culture F Culture G Culture H
medium
SB→SB Culture I Culture J Culture K Culture L
1x (1 mg/vitamin/L) and 10x (10 mg/vitamin/L) refers to the amount of vitamins added to the defined
medium. SB was super broth medium. One milliliter samples were taken at the following times post
induction: 1 hour, 3 hours, 5.5 hours and 24 hours.
was injected into each of 12 culture tubes containing 3.5 ml of the predetermined
conditions (four in defined medium, four in defined medium enriched by 10x vitamins
50
and four in super broth) as shown in Table 3.2. These twelve small cultures were grown overnight. One ml of each culture was saved for testing. 2.5 ml of each culture was added to 22.5 ml of the medium of choice at room temperature for a total volume of 25 ml. See Table 3.2 for the order of addition of IPTG and/or DAP to the various cultures.
3.3.8 Comparative expression of three mutants in two E. coli strains. A comparative study, on the growth of rFab2, rFab3 and rFab 4 transformed into either E. coli BL21 (DE3) [DE3] or E. coli BL21 (DE3)-RIL [RIL] cell lines, was undertaken to determine the preferred cell line for optimum yield in flask culture. Matched baffled flasks were purchased from Daigger. Twelve cultures were set up on one day and another six cultures were done the following day with the same stock cultures grown to the same O. D. 600 nm. Protein induction followed the same procedure outlined in section 3.3.3 but the O. D. 600 nm of each inoculum was normalized to the same value by the addition of defined medium as needed. Samples were taken for O.D 600nm and
ELISA measurements throughout the process. To relate the O.D. 405nm used for ELISA samples to protein concentration, purified rFab standards of known concentration were included on each ELISA plate and the O.D. 405nm readings for each culture were compared with the standards.
3.4 Results
3.4.1 Cloning of mouse recombinant Mab25.3 and modifications of the pET28 plasmid. The completed pCOMB3H plasmid (pCOMB-Fab1) did not express well in E. coli. Since the original heavy and light chains isolated from the Mab25.3 mRNA were not well expressed, engineering the rFab further to improve its expression
51
was undertaken by Dr. Momany. With the goal of also including a purification handle with it in the form of a His tag, Fab1 was modified by insertion of a 6-His tag on the C terminus of the heavy chain. Figure 3.2 shows the genetic organization of the pET28-Fab
S B I G T R A SacI O T7 m pA * *
* f a b L
r
n XbaI a TAA K RIBS
P ATG
e
l
B
*
XhoI
*
f
* a
b H
o ri lacI SpeI TAA
pET28-Fab
Figure 3.2: Insertion of the “fab” Genes into pET28 Vector. Abbreviations are:
T7, T7 promoter; RIBS, ribosome binding site, lacI is the lactose (IPTG) response
r gene, Kan , kanamycin resistance gene; fabL, Fab light chain; fabH, Fab heavy chain;
ori, origin of replication. ATG is the start codon and TAA is the stop codon. OmpA
and PelB are leader sequences directing the fragment to fold in the oxidative
environment of the periplasmic space. See Appendix C.
dsDNA plasmid with light (fabL) and heavy chain (fabH) insert is shown. Each inserted gene had its own ribosome binding site binding site (RIBS) for proper translation. OmpA
52
and PelB were leader sequences that targeted the protein to the periplasm. A kanamycin
resistance gene (Kanr) was used for selection for the E. coli clone containing the mutant.
The translated polypeptide sequence that resulted from this mutation of the heavy chain is
shown in Figure 3.3.
Vicinal histidines can also interact with metal chelate columns as an alternative to the 6-His tag of rFab2. To accomplish the vicinal histidine configuration of rFab3 a deletion primer was designed to remove 12 residues and add the threonine and serine residues after the two retained histidines on the C-terminus. By multi-site mutagenesis,
4 of the 6 histidine residues on the heavy chain C-terminus were deleted and the light chain also mutated utilizing a second PCR to produce 2 histidine residues on the light chain C-terminus. To mutate the glutamate back to a glutamine only required a single base modification at the N-terminus. For rFab4 the deletion of the superfluous leucine required the design of a deletion mutation primer. For rFab5 a silent mutation (Van 91 I restriction endonuclease site) was introduced in addition to the N57→K mutation.
As shown in Figure 3.4 , DI to EL residue changes in the light chain were conservative changes since isoleucine and leucine have the same hydrophobicity and aspartate and glutamate both carry a negative charge with only one CH2 group difference
in their size. These conservative changes to the light chain introduced a Sac I
endonuclease restriction site. The only other mutation to this chain was on the N-
terminus where two histidine residues were added to become vicinal (nearest neighbor)
residues to the histidine residues inserted on the N-terminus of the heavy chain.
After studying the structure of the monoclonal Fab25.3 (1AFV), an unplanned leucine insertion mutation to the heavy chain in a surface β-pleated sheet was discovered
53
QVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGEIHPNSGNTNYNEKFK
GKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARWRYGSPYYFDYWGQGTTLTVSSAKTTPPSVYP
LAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTW
PSETVTCNVAHPASSTKVDKKIVPK
Heavy Chain Recombinant Mutants
rFab2 EVQLL–QPG------G57NT------KKIVPRDCGVTSHHHHHH# rFab3 QVQLLEQPG------G57NT------KKIVPHHTS# rFab4 QVQLEQPG------G57NT------KKIVPHHTS# rFab5 QVQLEQPG------G57KT------KKIVPHHTS#
Figure 3.3: Recombinant Mutants of the Heavy Chain. The original sequence of
the monoclonal Fab is at the top of the figure. Highlighted sequences were areas
subject to mutations. The insertion of an extra leucine residue was not intentional but
was discovered by comparing the sequence with the monoclonal Fab sequence in the
PDB. Modifications to the sequence are highlighted by violet, blue and red text. #
stands for the stop codon TAA
that had been carried over from the original pCOMB vector. Mutation primers
HCREVDELL and HCFORDELL were utilized to rectify the unfavorable leucine insertion creating rFab4. This mutation decreased the number of HPLC peaks to one.
54
DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNLGSGVPAR
FSGSGSGTDFSLNIHPMEEEDTAMYFCQQSKEVPLTFGAGTKVELKRADAAPTVSIFPPSSEQLT
SGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNS
YTCEATHKTSTSPIVKSFNRNE
Light Chain Recombinant Mutants
rFab2 ELVLTQSP------SFNMN#
rFab3 ELVLTQSP------SFNMNHH#
rFab4 ELVLTQSP------SFNMNHH#
rFab5 ELVLTQSP------SFNMNHH#
Figure 3.4: Recombinant Mutants of the Light Chain. The original sequence is at
the top of the figure. Modifications to the sequence are highlighted by blue text. See
text for discussion of the different mutants.
Mutant rFab5, designed to improve crystallization properties of the rFab, had an
(N57K) mutation of the heavy chain with an adjacent silent Van 91 I endonuclease restriction site engineered into the DNA to distinguish these clones from the rFab4 mutant. The mutagenesis was accomplished with the use of primers FAB5FOR and
FAB5REV. The side chains required a bivalent cation for salt-bridging interactions in the native Fab25.3 crystal form. The glutamine in the heavy chain could not be mutated
55
in the library crFab to a lysine residue to guarantee formation of the salt-bridge because
this increased its sensitivity to protease recognition and degradation.
The N-terminal sequence had another unplanned sequence mutation in it that
changed a surface glutamine to glutamate increasing the number of negative charges on
the surface of the rFab by one negative charge. Since the lysine mutation was supposed
to salt-bridge with an aspartate on another Fab molecule within the crystal lattice, this
could cause heterogeneity in the orientation of the molecules within the lattice by
providing alternating salt-bridge type interactions. (See crystallization results 3.4.5.)
3.4.2 Expression and purification of recombinant Fab. The original rFab2
mutant was poorly expressed in pCOMB3H, but transfer of the DNA to the pET28
plasmid improved its expression in E. coli.
Shown in Figure 3.5 panel A, HPLC Zn chelate column results for a 1-liter prep
of precipitated defined medium containing mutant rFab3. Panel B shows a Zn chelate
column separation of periplasm extract for the same 1-liter cell prep. In both panels the
gradient was formed between buffer A1 = 0.5M NaCl, 0.02 M sodium phosphate, pH 7.4,
and buffer B1 = A1 + 0.5 M imidazole, pH 7.4. The relative heights of the two peaks were similar. Although the peak in Figure 3.5 panel B represents about 5 mg of protein it is only about 50% rFab3 at this stage of purification. Indication that it was not pure was shown by the height of the UV 340nm and UV 412nm peaks under it. Purified rFab has no 340 nm or 412 nm signal. There was more rFab3 protein in this peak from defined
medium, and the defined medium was cleaner than the periplasm extract.
In Figure 3.6 the further purification of expressed rFab3 mutant demonstrated
difficulties with this protein. The follow-up Q column results for the previous Zn chelate
56
column purification of proteins demonstrated the heterogeneous nature of rFab3. The
variability of the structure was evidenced by multiple small HPLC peaks. They were differentially released from the Q column. The six peaks between 50 and 100 ml on the
top panel for periplasm extract were all rFab3 peaks and gave strong ELISA signals. The
remaining peaks were E. coli proteins. The bottom panel showed a similar number of rFab3 peaks from the supernatant extract but the proportions were different. Hetero-
geneity was obviously a problem whether it was extracted from the periplasm or the
supernatant. What this did show was that there were fewer interfering E. coli proteins in
the supernatant and that there was a quantitative advantage as well since more protein
could be extracted from the supernatant than from the periplasm.
The yields, shown in Table 3.3, for cultures grown under similar conditions were determined from the integrated peak for the coupled Q Ni NTA column purification, except for rFab3. The rFab3 yield is taken from the highest peak out of 5 ELISA positive
peaks shown in the graph of Q-column purification peaks in Figure 3.6. The (HC N57K) mutation produced a much poorer yield of rFab5 protein per liter than the rFab4 mutant.
See Figure 3.7 for the comparison of HPLC purification results between rFab4 and rFab5. Panel A shows the purification of 6 liters of defined medium containing rFab4 with a peak that contains 4.7 mg of purified rFab4. Panel B shows a 1 liter preparation of rFab5. The peak circled was not fully representative of the load. This run was restarted after a momentary delay.
The modified protein purification method described in section 3.3.4.2 was developed to separate the E. coli High-His protein from the vicinally His-tagged rFab.
The high-His protein composed nearly half of the protein isolated from periplasm with
57
A. UV1_280nm UV2_340nm UV3_430nm Cond% Concentration pH Inject UV1_280nm@BASE Conductivity
mAU 84.03 mS/cm
1000 100
500 50
0
0 0 50 100 150 ml
B. UV1_280nm UV2_340nm UV3_412nm Concentration Inject Conductivity pH
mS/cm mAU 56.0
54.0 3000
52.0
2000 50.0
48.0 1000 46.0
0 0 50 100 150 200 250 ml
Figure 3.5: HPLC rFab3 Purification Results for Defined Medium versus
Periplasm. In both panels the gradient was formed between A1 = 0.5M NaCl, 0.02 M
sodium phosphate, pH 7.4, and B = A1 + 0.5 M imidazole, pH 7.4.
58
A. UV1_280nm UV2_340nm UV3_430nm Conductivity Cond% Conc pH UV1_280nm@01,BASE
ELISA positive peaks
mAU mS/cm 55.25 200 60.0 150 104.84 121.87 100 40.0 156.96 63.83 91.50 50 20.0
0 0.0 0 50 100 150 200 ml Q Column Purification of rFab3 Periplasm
B. UV1_280nm UV2_340nm UV3_430nm Conductivity Cond% Conc pH UV1_280nm@01,BASE
ELISA positive peaks 44.61 mAU mS/cm 250 100 200 80 150 60 100 74.61 140.20 40 50 56.93 86.55 96.62 20 0 0 0 50 100 150 ml Q Column Purification of rFab3 Supernatant
Figure 3.6: Q Column Purification Results for rFab3 from Periplasm and
Supernatant Fractions from the Same 1-Liter batch.
59
A UV1_280nm UV2_340nm UV3_430nm Conductivity Cond% Conc pH UV1_280nm@01,BASE
mAU mS/cm 3500 800 3000 E. Coli 14 KDa 600 2500 high-His protein Fab4 2000 417.07 400 205.20
1500 200 1000 0 500 -200 0 Waste 0 100 200 300 400 ml
Coupled Q-Ni NTA Column Purification Scheme
B UV1_280nm UV2_340nm UV3_430nm Conductivity Cond% Conc pH UV1_280nm@01,BASE
389.25 mAU mS/cm
500 80.0 400 60.0 300 353.99 200 Fab5 40.0 304.23 100 142.80 323.71 255.33275.00 20.0 - -2.21 0 Waste 0 100 200 300 400 ml
Coupled Q-Ni NTA Column Purification Scheme
Figure 3.7: Comparison of HPLC Purification for rFab4 and rFab5. Panel A was
the purification of rFab4 from 6 liters of defined medium by coupled Q-Ni NTA
columns and panel B was a similar purification of rFab5 from 1 liter of medium. See
section 3.3.4.2 for details of HPLC purification.
60
the His-tagged crFab when only a nickel or zinc chelating column was used (Figure 3.5).
By switching from a 20 mM phosphate-buffered, 0.5 M NaCl solution, pH 7.4, to a 20 mM tris HCl- buffered, 0.1 M NaCl solution, pH 7.4, and incorporating a Q column ahead of the nickel chelating column, it was possible to separate the High-His protein from the vicinal His-tagged crFab. The High-His protein bound tightly to the Q column until eluted with the high NaCl solution (buffer A12). The rFab came off the nickel- chelating column early in the gradient in the presence of low salt and imidazole. This step eliminated all but trace E. coli proteins. However, these trace proteins cannot be ignored because there was presumptive evidence that at least some of them were proteases that, given enough time at 4oC, would digest the crFabs (no ELISA signal from samples stored at -20ºC after a few months in the freezer).
Thiophylic adsorption chromatography has been shown to be selective for immunoglobulins via a salt promoted process at neutral pH. Adsorption is promoted by anions and cations of the Hoffmeister series that are counter to the chaotropic ions.
Ammonium and potassium sulfate work well with immunoglobulins (Hutchens & Porath,
1986). It has also been used to separate the Bence-Jones dimer from the correctly associated rFab heterodimer (Fiedler & Skerra, 1999).
As shown in Figure 3.8, the HPLC separation on the T-gel column was accomplished manually. Prior to loading the defined medium salt concentration was brought up to 1.0 M ammonium sulfate by addition of solid ammonium sulfate. The direct purification of 500 ml defined medium containing rFab4 from the defined medium utilized a T-gel matrix packed in a Pharmacia Biotech XK-16 column and loaded onto the column at a flow rate of 10 ml per minute. The load was stopped and restarted and the
61
gradient was started after the mAu 280 reached a relatively flat baseline. The automated
zeroing of the HPLC instrument did not work well in the manual run mode and thus the
apparent peak below zero is not real. In Figure 3.8 there was no evidence of a Bence-
Jones dimer peak for rFab4. The only peak was the heterodimer peak representing about
17 mg of rFab protein. Direct purification eliminated the 8-hour or longer precipitation step and the 8-hour dialyses steps to reduce exposure of the rFab protein to proteolysis.
UV1_280nm UV2_360nm UV3_480nm Cond Cond%
Conc pH Inject UV1_280nm@BASE
mAU mS/cm
120 2000 100
80 1000 60 0 553.87 40
20 -1000
-500 0 500 ml
Figure 3.8: HPLC Results for T-gel Purification of rFab4 from Defined Medium.
The separation was accomplished by a gradient between buffer A = 1.0 M ammonium
sulfate in 50 mM Na Phosphate, pH 7.5, and buffer B = 50 mM Na Phosphate, pH 7.5.
62
3.4.3 Physical characteristics of rFab2, rFab3, rFab4 and rFab5. The protein molecular weights, net formal charge, total number of amino acids, molar absorbance and yields are reported below in Table 3.3. The molar absorbance was calculated and was the same for all three because there was no change in the number of cysteines, tryptophans or tyrosines. The web site for calculating these as well as DNA and RNA physical constants is the following: http://paris.chem.yale.edu/cgi-bin/. A 1 cm light path was assumed.
The between rFab4 and rFab5 and Table 3.3 for a comparison of yields. If this had been
the only problem we could have scaled up the production as needed, but it crystallized
poorly as shown in Figure 3.12.
Table 3.3 Physical Characteristics of rFab Mutants.
Mutant Calculated Total Number Molar Formal Yield per Liter
Molecular of Amino Absorbance Charge of Culture
Weight (Da) Acids (M*cm)-1 Supernatant
rFab2 48829.8 450 86735 -1 0.6 mg/L
rFab3 47956.8 442 86735 -2 1.8 mg/L
rFab4 47852.6 441 86735 -2 0.8 mg/L
rFab5 47866.7 441 86735 -1 0.2 mg/L
The calculated molecular weight used average isotopic mass numbers. The Molar Absorbance was
calculated using the method of von Hippel where ε = 5690 * (# of tryptophans) + 1280 * (# of tyrosines) +
6* (# of cysteines). 1 Absorbance unit (O.D at 280 nm) equaled 0.553 mg/ml. The heavy chain (N57K)
mutation produced a much poorer yield of rFab5 protein per liter than the rFab4 mutant. See Figure 3.5 for
the comparison of HPLC purification results
63
3.4.4. Native and SDS-PAGE gels results. A study was done of a native gel utilizing Fab3 and Fab5 in combination with its recombinant p24 antigen. See Figure 3.9 for evidence of the interaction between rFab3 and rFab5 with p24. The broad bands in the p24 + rFab3 and p24 + rFab5 lanes were indicative of the interaction between these proteins, with a corresponding decrease in the individual protein bands. There were no
p24 Fab5 p24 + Fab5 p24 + Fab3 Fab3
Figure 3.9: Native Gel rFab Proteins with p24. This gel is on a precast 12.5% Phast gel using native buffer strips. These rFab proteins ran at the size of the folded proteins. P24 alone had multermeric bands as did rFab3 and rFab5. The rFab3 plus p24 shows a broad band with obliteration of the main rFab3 peaks. The rFab5 + p24 lane shows a broad banding and shifting of the center of the band approximately where the p24 peak should have been with decreased rFab5 peaks.
64
protein standards for this type of PAGE gel. The lanes where rFab5 or rFab3 were
combined with HIV I capsid p24 target showed a smeared band indicating association with the target to varying degrees. One may speculate that the broad-bands were caused by poorly-defined associations between these molecules that led to the poor crystal forms for rFab3 and rFab5 seen in Figure 3.12.
Figure 3.10 contained a series of samples from various purification steps for rFab3. Lane 1 is defined medium from the inoculum. Lane 2 is the periplasm fraction after the removal of cell debris by centrifugation. Lane 3 is the periplasm fraction after ammonium sulfate precipitation concentrated the proteins. Lane 4 is the periplasm extract after dialysis that showed the selective removal of some E. coli proteins. Lane 5 is the purified HI-His protein form E. coli. Lane 6 is straight defined medium after debris
by centrifugation. Lane 3 is the periplasm fraction after ammonium sulfate precipitation concentrated the proteins. Lane 4 is the periplasm extract after dialysis that showed the selective removal of some E. coli proteins. Lane 5 is the purified HI-His protein form E.
coli. Lane 6 is straight defined medium after removing the cells. Lane 7 is ammonium
sulfate precipitated proteins from the defined medium before dialysis. Lanes 8 and 9 are
Q column fractions of the rFab peak. Lane 10 is the purified rFab4. Although there was nothing in the literature about the E. coli protein purified in lane 5, I have heard it called the “High-His protein” by those in the industry who purify proteins from that source for commercial and research purposes. It r was separated from the His- tagged recombinant
Fab protein by negatively-charged Q-sepharose columns and not by metal-chelate column
separation. The heavy and light chains were separated by high density gels as shown in
lanes 8, 9 and 10.
65
1 2 3 4 5 6 7 8 9 10 11
KDa 94 67 43 30 21 14
Figure 3.10: Purification of rFab4 from Periplasm and Defined Medium. A Phast precast HD SDS-PAGE gel demonstrated various steps of the purification process.
LMWM were low molecular weight markers of 14 KDa, 21 KDa, 30 KDa, 43 KDa,
67 KDa and 94 KDa molecular weight proteins as indicated in lane 11.
In Figure 3.11, the purity of the rFab4 peak is demonstrated after a single purification run of 1 L of defined medium extract using the piggybacked Q-Ni-NTA
Chelate column configuration and three buffers demonstrated. The 14 kDa E. coli protein is still evident in lanes 2 and 4 although significantly reduced. The Q column was probably overloaded so that a trace amount of the High His protein also bound to the Ni chelate column.
66
t
n
i
n
e
a
t
t
o
a
r
n
p
r
5
3
e
d
1
1 3 5
p
e
t 1 1
u
k
k
a
s
M
M
t
p
p k k
i
d p p
W 4
p W 4
e
i
t
b
4 b 4
c
M
M
a
a
u
b b
e
l
a a
F L
L r F
i
p F F
d
x x
x x
n
0 5
0 x x 0 x
1 1
U 1 5 5 1 5
94 67 43
30
21
14
Figure 3.11: High Density SDS-PAGE Gel of rFab4 Mutant. 5x, 10x and 15x refer
to the concentration factors for the concentrated rFab protein (5x is less concentrated
than 10x). The undiluted supernatant showed evidence of being enriched for the
rFab4 protein. Abbreviations used: LMWM; low molecular weight markers, 10x and
15x were dilution factors for the markers.
3.4.5 Crystallization results. Crystallization results for the uncomplexed mutants were the lynch pin on which the “go” or “no go” decisions were made for the mutant of choice. If the mutant could not crystallize in a reasonably short period of time
without the presence of its cognate antigen, it was assumed that it would not crystallize
any better in the presence of its cognate antigen. For a high-throughput crystallization
technology to be developed, it hinges on rapid crystallization of the proteins of interest.
Crystallization results are shown in Figure 3.12.
67
The well solutions that produced crystals were based on the solution that
produced the monoclonal Fab crystals of Fab25.3 with p24, namely 16-20% PEG 4000 in
100 mM bistris·HCl, pH 7.0. The few conditions found with the 1536-well screen were not reproduced. A few crystals were found with the Crystal Screen I kit crystallizations.
Mutant rFab4 formed highly twinned thin plate crystals (sheaf bundles) in the presence of
1.0M Li2SO4 and 12-25% PEG 4000. See Figure 3.12 for photos of crystals and crystal
data.
The pET28-Fab2 protein did not crystallize for Dr. Momany. The rFab3 mutant
also had very poor crystallization characteristics. The vicinal His mutations did no produce immediate crystallization results. After 10 months two tiny needle crystals closely associated with each other appeared in one drop. rFab4 crystallized after only two to three weeks. Although the crystallization properties of rFab4 were not stellar, they were sufficient to make it the choice for the basis of the co-crystallization library to be detailed in Chapter. The rFab4 crystals appeared after two to three weeks and were well formed, albeit small, orthorhombic crystals. X-ray data were collected on a local rotating-anode X-ray source (CuKα). The Fab3 needle crystal was X- rayed but was too small to determine cell constants or space group. The rFab5 crystal was too badly twinned to try to obtain any data. The badly twinned crystal of rFab5 hinted at another problem with the DNA sequence. The N-terminal sequence had another unplanned sequence mutation in it that changed a surface glutamine to a glutamate increasing the number of negative charges on the surface of the rFab by one negative charge. Since the lysine mutation was supposed to salt-bridge with an aspartate on another Fab molecule within the crystal lattice, this could cause heterogeneity in the orientation of the
68
rFab4 Orthorhombic Crystals
2-3 weeks to crystallize
Size 0.22 x 0.15 x 0.04 mm
The Space Group was C222 or C2221
Unit cell parameters:
a = 73.58 Å, b = 98.57 Å and c = 126.38 Å
They diffracted up to a resolution of 5.25 Å.
Needle Crystals of rFab3 Badly Twinned Crystal of rFab5
10 months to crystallize 6 months to crystallize
Diffracted to 7 Å
Figure 3.12: Crystals of rFab3, rFab4 and rFab5 Proteins. Color effects were
due to plane polarized light. All crystals were photographed at the same level of
magnification.
69
molecules within the lattice by providing alternating salt-bridge type interactions.
Compared with the other two rFabs that crystallized as shown in Figure 3.12, rFab4 definitely was favored because of its single crystal habit and relatively larger size.
3.4.6 Diaminopimelate effect on expression. In Figure 3.13 the graphs showed a general trend that diaminopimelate had little or no effect on protein induction and expression to the medium. It may have had an enhancement effect on periplasm expression. There was little or no constitutive expression of crFab in cultures A or E or in the diaminopimelate alone induced cultures B and F. There was a similar amount of rFab produced to the defined medium whether diaminopimelate was there or not in the remaining cultures. The data were expressed in O.D. 405 units (10x dilutions) and there was no box-to-box scaling for these measurements. Therefore the cell-wall cross-linking reagent probably had no effect on rFab production to the medium, although it may have had an enhancement effect on production to the periplasm. Without replicate data it can not be stated as a fact. These experiments were not repeated. Other experiments showed variable amounts produced in super broth, but these results might lead one to believe that rFab was not expressed in super broth medium. Since the positive controls did not work on that tray the results were uninterpretable. However, they did guide us away from using super broth and toward taking advantage of the defined medium. Defined medium is a much cleaner medium to work with because it does not start out contaminated with any cellular proteins. The only proteins that came into the medium were those that had been expressed by E. coli cell metabolism or breakdown. Localization of the rFab protein to the periplasm or the medium was more dependent on the strain of E. coli than
70
A
ELISA Signal of Supernatant rFab3 Cultures 4.0
3.5 3.0 Overnight
2.5 1hr
2.0 3hr
1.5 5.5hr O.D 405 nm 405 O.D 1.0 24hr 0.5
0
ABCDE FGH I JK L
Culture
B
ELISA Signal of Periplasm of rFab3 Cultures
3.5
3.0 Overnight 2.5 1 hr 2.0 3 hr 1.5 O.D. 405 O.D. 5.5 hr 1.0 24 hr 0.5
0 AB CDE FGH I J K L Culture
Figure 3.13: Diaminopimelate IPTG Induction of pET28-Fab3. Cultures A through D were started in defined medium + 10x vitamins and then switched to defined medium 1x vitamins. Cultures E through H were maintained in defined medium +1x Vitamins and Cultures I-L were maintained in super broth medium.
71
the medium itself. No attempt to correlate the O.D. 405nm data with protein
concentration was made.
3.4.7 Six-culture comparative expression of three mutants. In Figure 3.14 the
results of the comparative study on the growth of Fab2, Fab3 and Fab 4 grown in either
E. coli BL21 (DE3) [DE3] or E. coli BL21 (DE3)-RIL [RIL] cell lines are shown. All cultures were done in triplicate. The data points were the averaged value for the three replicates. In Figure 5.3 panel A, the RIL cultures had a small competitive growth advantage over the DE3 cultures in sustainable growth for six hours after induction. The cultures were able to maintain an O.D. 600 nm reading of about 6 as compared with the
DE3 cultures reading of about 5. To relate O.D. 405nm to protein concentration in
Figure 5.3 panel B, rFab standards of known concentration were included on each ELISA plate and the O.D. 405nm readings for each culture were compared to the standards.
Utilization of baffled flasks improved aeration of the cultures and probably led to a quicker exhaustion of available nutrients for protein production peaking at about 4.5 hours post IPTG induction (Figure 3.14 panel B). The induction period should have been shortened to about 30 minutes after inoculation into the larger culture in order to catch the cells in early log phase of growth. It could be expected that if the cultures had been carried on for a longer period of time the E. coli cultures would have cyclically reutilized nutrients in the medium as cells lysed and new cells grew beyond the6-hour post-IPTG induction point [as evidenced by the 24-hour response of the culture in Figure 3.13].
While fairly high mg L-1 quantities could be obtained with the Fab2 and Fab3 mutants, it
was particularly difficult to predict when Fab3 would attain its peak mg L-1 value--
72
A. IPTG Induction 8.0
7.0
6.0 Fab2DE3 5.0 Fab2RIL Fab3DE3 4.0 Fab3RIL O.D. 600 O.D. 3.0 Fab4DE3 Fab4RIL 2.0
1.0
0.0 -3.0 -1.5 0 1.5 3.0 4.5 6.0 Time in Hours
B.
3.0
2.5 Fab2DE3 Fab2RIL 2.0 Fab3DE3
1.5 Fab3RIL
mg/L Fab4DE3 1.0 Fab4RIL
0.5
0 0 1.5 3 4.5 6
Hours Post-IPTG Induction
Figure 3.14: Growth of E. coli and Production of rFab Mutants. Panel A is growth of E. coli cultures expressed in O.D. 600 nm units. Panel B is mg/L rFab protein production estimated by ELISA from the standard concentration of rFab4.
73
usually sometime between 3 and 4 hours. With this experiment, rFab3 produced more product in the DE3 cell line than in the RIL cell line but this was not consistent with other earlier experiments. Its unpredictability made it a particularly difficult mutant to estimate the optimum time for harvesting the medium.
3.5 Discussion
Since we found a lot of rFab in the defined medium we initially assumed it was possibly leaking out of the cells due to an osmotic problem and thus decreasing our overall yield. However, that was not the case. When we tested the induction of protein with and without a cell-wall cross-linking metabolite, it essentially had no effect on the amount of induced protein that was secreted in the medium. Diaminopimelate had a small effect on the amount in the periplasm fraction. Considering that the rFab in the medium is more homogeneous, it was apparent that it would be preferable to extract the rFab from the medium. Periplasm fractions contain a mixture of Fabs because the PelB and OmpA leader sequences are in varying stages of being removed from the completed product. Once the folded Fab leaves the periplasm and is either excreted or leaked into the medium, it is folded, associated as a dimer and fully trimmed of the leader sequences.
Thus it is a much more homogeneous product than the periplasm fraction
Rapid purification to homogeneity was considerably improved by switching to the coupled Q-Ni columns approach. Direct purification of the rFabs from the medium using a T-gel column looks promising since addition of ammonium sulfate and running the
HPLC instrument can be done on the same day that the proteins are produced without waiting 8 hours for the proteins to precipitate.
74
The production and poor crystallization of Fab5 was a disappointing result.
Antibody fragment engineering can only go so far. Since E. coli digests proteins for its own metabolic requirements, problems like the ones we encountered are to be expected.
If the crystallization had worked well, then the problem of lower yield might have been overcome by optimization of production in a bioreactor. The broad bands in the native gel highlight a problem that plagues co-crystallization, namely poorly defined interactions between two molecules that may lead to disorder. More sharply focused bands in the native gel interaction between the target and the rFab, significantly displaced from the target protein and the rFab bands, could serve as an indicator of how well they might co-crystallize.
75
CHAPTER 4
MOUSE RECOMBINANT ANTIBODY FRAGMENT PRODUCTION IN A
BIOREACTOR
4.1 Overview
Two bioreactor processes utilizing the E. coli BL21 (DE3)-RIL strain (RIL strain) containing the pET28 rFab4 mutant were conducted and the results reported. E. coli
BL21 (DE3)-RIL is a low protease strain of bacteria. As described previously, the pET28b plasmid was engineered by Novagen to have a T7 promoter site and specific endonuclease restriction sites appropriate for the insertion of our mutant. The pET28-
Fab4 was IPTG induced according to a protocol based on bioreactor methods. The maximum amount of protein produced in flask culture for the rFab4 protein in the pET28 plasmid was 2.6 milligrams per liter (mg/L) (Kelley & Momany, 2003, #1). The maximum amount of rFab4 protein is yet to be realized, although 10 mg/L was evidenced by ELISA from the first Bioreactor process to be reported here.
4.2 Rationale
A bioreactor is an enclosed chamber in which biological reactions can occur and is equipped with various sensors to detect important life sustaining parameters for the contained culture (Peddie et al., 1996). Our rationale for utilizing the bioreactor was as
76
follows. The bioreactor process allowed for a much more finely tuned process than the shake-flask fermentation results shown in Figure 3.1. It offered a greater degree of control of parameters like glucose feeding, dissolved oxygen concentration and pH by the addition of appropriate amounts of either acid or base. Probably the most important differences between shake flask and a bioreactor are higher oxygen transfer into a bioreactor and the ability to control pH in the bioreactor. Since there may be a greater degree of control over all of the parameters in the experiment, a much higher ratio of product per volume of culture is achievable than in the shake-flask culture. An approximate measurement for observing cell growth in liquid culture is obtained by measuring the optical density of the culture at 600 nm (O.D. 600). The maximum O.D.
600 for flask cultures is about 5, but for bioreactors it can easily go as high as 40 depending on how the parameters are set. A higher cell density means more cells to induce product. Under optimized conditions a 1-liter culture in a 1.5-liter bioreactor is sufficient to produce milligram to gram quantities of rFab product (Chadd & Chamow,
2001, Leibiger et al., 1995).
4.3 Materials and Methods
Kanamycin, chloramphenicol, isopropyl-β,D-thiogalactopyranoside (IPTG), ethylenediaminetetraacetic acid (EDTA), ammonium sulfate, phenylmethylsulfonyl fluoride (PMSF) were purchased from Fisher Scientific. Vitamins and L-amino acids were a gift from Michelle Momany. Buffer and reagent chemicals were of the highest analytical or molecular grade available from commercial vendors. The defined medium was prepared according to the previous protocol (Appendix B) with the minor
77
modification being that the 860ml of water and 100 ml of 20x M9 solution were autoclaved within the bioreactor itself. The autoclaved glucose solution and the sterile- filtered solutions (amino acid mixtures, iron sulfate and vitamins) were added via a presterilized funnel through a sterile port on the head plate of the bioreactor. Antibiotics were injected using a needle and syringe through a needle port as was the 0.5 ml of 1 M
IPTG at the appropriate time for the induction.
The pH and DO probes were calibrated and all parts of the 1.5 L bioreactor were assembled and autoclaved along with the diluted M9 salts as stated above. The pH was maintained by the pH analyzer. It calculated the amount of acid or base to add to the medium and sent commands to the acid/base pump which incrementally added acid or base to the medium. Glucose concentration was controlled by on-line analysis using a glucose analyzer, which withdrew a 1-2 ml sample from the culture every 20-30 minutes.
The analyzer calculated the amount of glucose feed solution to add based on the volume of bioreactor contents and the current concentration of glucose. A temperature probe monitored the temperature. The temperature was controlled by continuous water flow through a circulation tube and a hot pad at the base. Aeration was achieved by sparging air at 1 L/min, controlling rotation (up to 1000 rpm) of a central shaft with two impellers and creating turbulence with baffles on the sidewalls. See Figure 4.1 for the actual setup of the first bioreactor process.
The first bioreactor process incorporated 20% HCl as the acid and 20% (10M)
NaOH as the base for maintaining the pH at the set point of 7.0. An initial growth period with glucose feeding of 8 hours helped to maximize the cell density. Nine hours prior to isopropyl-β-D-thiogalactopyranoside (IPTG) induction of the culture, 100 ml of
78
DO & pH Probe readings
Glucose Analyzer
Sampling port
Figure 4.1: Bioreactor Process Utilizing on-Line Glucose Feeding. The red arrow points to the acid intake and the blue arrow to the base dispensing lines that were set up in front of the pH controller and pumps. The bioreactor vessel contained the culture. The orange arrow points to the sampling port used for taking intermittent samples (for pH, ELISA results etc.). Intake and outlet ports located on the head plate of the bioreactor included a port for the dissolved oxygen (DO) sensor and the pH probe. The DO and the pH probes were calibrated. The entire assembly, including the
DO and the pH probes, along with the water for the defined medium was sterilized prior to use. Geoffrey Smith helped me set up the first bioreactor process and calibrated the DO and pH probes and Sarah Lee set up and calibrate the Biochemistry
Analyser YSI 2700 Select glucose analyzer for both processes.
79
inoculum was added to the defined medium in the bioreactor. The pH was monitored
both by visual inspection of the pH on the instrument monitor and by taking intermittent
samples and checking the measurement with an external pH meter that had also been standardized. The temperature of the culture was cooled after 8.0 hours from 37ºC to
25ºC over a period of about 20 minutes and then after another 40 minutes, IPTG was injected into the culture to a final concentration of 500 µM to begin IPTG induction. The culture was maintained at 25ºC for the remainder of the experiment. Sampling was every hour after the induction phase was begun.
The O.D. 600 was measured on a spectrophotometer using a 20-fold dilution of the culture sample in water and blanked against water. The concentration of rFab4 was determined relative to Fab4 ELISA standards and does not exclude detection of light chain dimers.
A second experiment was conducted with E. coli BL21(DE3)-RIL culture containing the pET28-Fab4 plasmid but NH4OH was substituted for NaOH as the titrant
for maintaining pH, but keeping all the other culture nutrient parameters the same. The
40% (3 M) NH4OH acted as the base for maintaining the pH at the set point of 7.0 and
was used as an alternate nitrogen source. The 20% HCl remained the same. None of the
other culture parameters were intentionally varied, but it was predetermined that we
would allow the culture to grow to an approximate O.D. 600 nm of 40 and assumed a
normal growth curve to take 24 hours. The inoculum was added at 3 p.m. The 50%
glucose solution used was taken from another student’s (Atin Tomar) completed
bioreactor process and inadvertently also contained 12% (w/v) sodium acetate. The
culture grew very fast by comparison to the first process. After only seven hours of the
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growth phase, glucose feeding was no longer necessary. At 10 p.m. the glucose feed was
stopped and cool-down was begun to 25ºC over a period of 60 minutes and then IPTG
induced in the same manner as before. Sampling was hourly during the growth phase and
after 11 p.m. every 1.5 hours during the post-induction phase because it occurred so late.
(A. Tomar withdrew four of the post-induction samples and recorded the O. D 600,
volumes of glucose, acid and base remaining and the pH for those time points. The final
time point measurements were made by the author of this dissertation.)
4.4 Results
Results for the first bioreactor process are shown in Figure 4.2 below. The optical
density (O.D. 600) climbed rapidly supported by glucose feeding and the excellent
aeration. Even after glucose feeding was stopped at IPTG induction the culture continued
to show sustainable growth until five hours post –IPTG induction. The drop after that is
usually indicative of a cycling pattern that occurs when a culture’s growth has been
synchronized. The pH was very well controlled throughout this experiment, 6.98-7.02.
The dissolved oxygen concentration was high and the appearance of the Fab4 product as detected by ELISA was remarkable. In Table 4.1 the amounts of acid and base used to control the pH during the intervening times was recorded. The sudden jump from 2.8 ml to14 ml of acid consumed in the one hour time period from 2 to 3 p.m. is regarded as the switch point to acetate metabolism as the carbon source by E. coli when the glucose in the medium has been exhausted.
“Foam out” is an expression describing a culture that has reached a high level of protein within the medium and the fast rotation of the impeller has whipped it into a
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foam. This foam often overflows the bioreactor vessel into a collection vessel. If caught before it overwhelms the vessel an antifoaming reagent can be used that immediately quells the foaming reaction. Foaming out of the first culture occurred some time in the night but no antifoaming agent was used. The amount of culture lost was approximated at 150 ml with 958 ml remaining at the end of the process.
IPTG Induction 14.0
12.0 %DO
10.0 100%
OD 600 8.0 80% pH mg/L Fab4 6.0 60% DO
O.D. 600, pH, mg/L pH, 600, O.D. 4.0 40%
2.0 20%
0.0 0% -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 Time in Hours Post IPTG Induction
Figure 4.2: First rFab4 Bioreactor Process. On the left side of the graph the units
could be read as pH (0-14) or mg/L Fab4 or the optical density at 600 nm. On the
right hand side the units are the dissolved oxygen (DO) as percentage of saturation.
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Table 4.1: First Bioreactor Fermentation Process O. D. 600 nm, % DO, Acid and Base Consumption over Time.
Hrs post -9 -1 0 1 2 3 4 5 6 IPTG
Time of 12 a.m. 8 a.m. 9 a.m. 10 a.m. 11am 12 p.m. 1 p.m. 2 p.m. 3 p.m. observation
O.D. 600 0.64 9.48 9.46 10.14 11.00 10.97 13.00 12.66 8.66
% DO 100 76.0 76.0 76.2 76.5 77.4 78.0 79.7 81.7
ml HCl 0. 85.0 5.0 4.0 6.3 0.7 0.2 2.8 14.0
ml NaOH 0. 75.0 7.0 3.0 2.5 2.5 6.2 0.3 0.5
The ml of acid or base is the milliliters of acid or base consumed during the time between data points. 118 ml of acid and 98 ml of base were consumed over the entire course of the experiment.
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The second bioreactor process results shown in Figure 4.3 were very different from the first. My notes indicated that I had accidentally used a glucose/acetate carbon source during the first three hours of the growth period. The presence of acetate apparently acted to buffer the medium so that a lot less acid and base were consumed in the growth phase as compared with the growth phase of the first bioreactor process apparently acted to buffer the medium so that a lot less acid and base were consumed in the growth phase as compared with the growth phase of the first bioreactor process shown in Table 4.1. Three hours after starting the process, the technician (Sarah Lee) discovered that the glucose solution was not what we had supposed. She measured the concentration of glucose, acetate and ethanol (by HPLC) from the sample that she took at
6 p.m. (4.1 g/L glucose, 1.0 g/L acetate, 3.2 g/L ethanol). The ethanol came from the chloramphenicol solution. E. coli preferentially uses glucose but will also use acetate as its carbon source in the absence of glucose. When the glucose carbon source was exhausted then E. coli switched over to acetate metabolism (Koplove & Cooney, 1978,
Lakshmi & Helling, 1978).
In Tables 4.1 and Figure 4.2, E. coli’s switching to acetate metabolism was detected by the increased use of HCl added to maintain the pH. 40% NH4OH seemed to over compensate the amount of base needed to maintain the pH at 7.0 because the pH was not as tightly maintained at 7.00 as in the previous experiment. However, the pH changes were not radical (pH ranged from 6.94 to7.02.) While the rise in O. D. 600 nm to a high of 37 may be remarkable in so short a time, the lack of production of Fab4 was disheartening (HPLC purification was negative for rFab peak and SDS-PAGE gel was negative for characteristic bands). E. coli protein production was probably up regulated
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to anabolic production of more cells and the production of proteases and other
nonessential proteins was down regulated.
Foaming was also a problem with the second bioreactor process. Adding 1 ml of an antifoaming reagent caused the DO probe to wildly fluctuate for about 2 hours until it stabilized again. The graph on Figure 4.3 could not show all of the fluctuations due to the limitations of the Excel program plot functions.
IPTG Induction
30
25 %DO
20 100% -30) -30) 14)
- O.D.600nm 15 75% pH
pH (0 DO
O.D.600 nm (0 nm O.D.600 10 50%
5 25%
0 0% -7 -6 -5 -4 -3 -2 -1 0 1.5 3 4.5 6 8 Time in Hours Post IPTG Induction
Figure 4.3: Second Fab4 Bioreactor Process. The ammonium hydroxide was at
40% concentration (vol/vol) (3M) and the HCl was at 20% (vol/vol) concentration.
Glucose feeding was stopped at induction.
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Table 4.2: Second Bioreactor Fermentation Process O. D. 600 nm, % DO, Acid and Base Consumption over Time.
Hrs post -8 -7 -6 -4. -2 -1 0. 1.5 3 4.5 6. 8 IPTG
Time of 3 p.m. 4 p.m. 5 p.m. 7 p.m. 9 p.m. 10.p.m. 11 p.m. 12:30 a.m. 2 a.m. 3:30 a.m. 5 a.m. 7 a.m. observation
O.D. 600 0.04 0.60 2.22 11.08 14.58 23.80 25.00 28.41 28.33 27.27 27.96 27.68
DO% 99.8 95.7 91.0 71.2 30.7 2.8 74.1 79.2 79.5 79.4 79.3 791
Acid (ml) 0 0.7 0.3 6.2 28.0 0 7.9 0.9 1.0 1.0 1.0 1.4
Base 0 2.0 2.0 6.0 19.6 9.9 0.5 1.0 0 0.5 0 0.8
The ml of acid or base is the milliliters of acid or base consumed during the time between data points. 48.4 ml of acid and 42.3 ml of base were consumed.
Since this process went much faster than normal the induction began at an unusual time of the day.
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4.5 Discussion
The two bioreactor processes described above were performed on the same cell
line and contained the identical plasmid. With the results from our first attempt at
producing Fab4, we were hopeful of solving the supply problem for obtaining enough
protein from a single process to do a complete crystallization screen consisting of 1536
different conditions with all necessary follow-up experiments. With the results from the
second process we learned that substituting NH4OH for NaOH might not be conducive to
the production of rFab4 protein. In the second bioreactor process, it is possible that the
presence of the ammonia turned off signaling for any protein production that was not directly involved in cell growth, but a lot of other problems occurred.
The inadvertent presence of acetate in the glucose feed leaves unanswered questions about why no protein was produced. Acetate has been reported to be toxic to
protein synthesis, but March et al., 2002, suggested by their research that “the rate of
acetate formation represents an inefficient consumption of glucose carbon, which is
reduced by the presence of pyruvate carboxylase,” stating “that acetate concentration
does not limit cell growth and protein synthesis, as predicted by other researchers.” An article by Farmer and Liao, 2001, stated that they were able to engineer E. coli to produce high levels of recombinant protein in the presence of acetate. They created a new induction system by reengineering the Ntr regulon. They inactivated phosphotrans- acetylase (pta gene product), which disrupted the acetate pathway and prevented it from synthesizing acetate. By addition of acetate they were able to obtain a high level of protein synthesis.
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In my opinion, the radical DO probe fluctuations in the second bioreactor process
did not reflect the actual dissolved oxygen level since oxygen flow to the bioreactor was
not concurrently interrupted. We did not have another method of detection of dissolved
oxygen. The sensor tip may have been coated by the surfactant blocking the passage of
oxygen freely through the sensor membrane until de-coating occurred over the time
frame of about 2 hours with the reestablishment of sensitivity. A study of oxygen sensing
in the presence of the dissolved surfactant alone may help sort out what was happening to
the DO probe when surfactant was added.
Various parameters for the induction of Fab4 production have not been optimized.
The IPTG concentration at induction has not been varied. Neither has the temperature drop for induction been optimized.
But regardless of the lack of optimization studies, preliminary studies showed that
production of mouse recombinant Fab4 protein in a bioreactor process may produce more
Fab4 protein, provided the parameters are correctly adjusted, than the amount that can be
produced in flask culture.
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CHAPTER 5
GENERATION OF A PHAGEMID MOUSE RECOMBINANT ANTIBODY
FRAGMENT LIBRARY BY MULTI-SITE DIRECTED MUTAGENESIS
5.1 Overview
Transfer of the rFab4 mutant from the pET28 plasmid into the pCOMB3H
phagemid was accomplished by a SacI/SpeI endonuclease excision of both the plasmid and the phagemid, gel purification of the excised rFab4 and linearized phagemid, and re- ligation of the DNA into pCOMB3H in frame with the gene III product to create the completed pCOMB-Fab4 phagemid.
A non-immune phagemid recombinant antibody fragment (rFab) library derived
from mouse monoclonal antibody fragment Fab25.3 to the HIV I capsid protein p24 was
generated directly from the pCOMB-Fab4 mutant. A nominal diversity of 1.0 x 107 was obtained utilizing multi-site directed mutagenesis technology based on modifications of
Stratagene's MultiSite™ Directed Mutagenesis kit. The library was expressed in the
XL1-blue strain of E. coli. Seven proteins available in the laboratory were selected for preliminary characterization of this library. Selection for protein-specific rFab antibodies and their amplification was effective after three panning cycles per protein for preliminary testing of the library. The percentages of colonies (out of a random selection of 25 colonies) that produced positive enzyme-linked immunosorbent assay (ELISA)
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signals to phagemid rFabs in the super broth medium ranged from 12% to 64%
depending on the protein tested. The use of multi-site directed mutagenesis allowed a
functional rFab library to be constructed in a very short time while retaining the structural
framework of the Fab molecule.
5.2 Rationale
Phagemid and phage libraries of various types of antibody fragments, [single
chain (scFv) (Jung et al., 1999 & Lou et al., 2001) and Fab antibody fragments (Soltes et al., 2003 & O’Connell et al., 2002)] are being generated both in industry for commercial
(Marks et al. 1991 & 1992) and medical uses (Cheetham et al., 1998, Chen et al., 1999,
Persic et al., 1997, Press et al., 1993 & Tuckey et al., 2002) and in academia for fundamental research purposes to determine interactions of molecules at the molecular level (Martineau & Betton, 1999, Kelley & Momany, 2003). The human genome project has spawned the “human proteome project,” the search for and characterization of all proteins encoded by the human genome that have not previously been discovered by conventional means. Other genome projects are also searching for insight into numerous aspects of structure and function at the molecular level for other biological species. We are developing rFabs as co-crystallization reagents as an alternative to conventional crystallization techniques used for determining the 3-dimensional structure of proteins.
As part of this goal, we have utilized multi-site mutagenesis as a rapid means of preparing a large library of rFabs while retaining a conserved structural scaffold. Fabs contain six complementarity determining region (CDR) loops, three in the heavy chain and three in the light chain, which recognize diverse antigens. In this experiment, the
90
third CDR loops of both the heavy and light chain were simultaneously randomly mutagenized with fixed length mutation primers. The remainder of the rFab had been optimized for efficient prokaryotic protein expression. Thus maintaining the integrity of
the remainder of the rFab was desired. By using multi-site directed mutagenesis, a
complex library was constructed in a minimal amount of time without requiring multiple
subcloning steps.
5.3 Materials and Methods
5.3.1 Fab4 DNA manipulation from pET28-Fab4 plasmid into pCOMB
plasmid and clonal selection for pCOMB-Fab4. The pET28b plasmid was purchased
from Novagen (Madison, WI). The restriction endonucleases Sac I, Spe I, Nhe I, Xba I and Xho I were purchased from New England Biochemical and ligase was purchased from Gibco. Ethylene diamine tetraacetic acid (EDTA) was from J. T. Baker and dehydrated LB broth, Miller LB agar, and isopropyl-β,D-thiogalactopyranoside (IPTG) were purchased from Fisher Biotech/ Fisher Scientific. Lysozyme and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma. The following DNA purification kits were purchased from QIAGEN: QIAquick Gel Extraction Kit,
QIAprep® Spin Miniprep Kit, QIAquick PCR Purification Kit and Plasmid Midi Kit.
Primers for the forward and reverse priming of the heavy and light chains were synthesized at the Molecular Genetics Instrumentation Facilities (MGIF), University of
Georgia, Athens, GA. Transformation of purified DNA into E. coli utilized a Bio-Rad E. coli Pulser and 2 mm gap cuvettes. See Chapter 3.2.3 for details on defined medium and
3.2.5 for the transformation protocol.
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Colonies were grown on freshly streaked LB agar plates containing the antibiotic
specific for the plasmid (kanamycin) and phagemid (carbenicillin) and then transferred to
LB broth and grown up overnight. The DNA was isolated and purified from each culture
using a Miniprep™ kit. Both the pCOMB3H phagemid DNA and the pET28-Fab4
plasmid DNA were purified by agarose gel extraction prior to transferring the Fab4 insert into the pCOMB3H phagemid. The pET28-Fab4 plasmid was endonuclease restriction
cut with Sac I and Spe I to prepare the Fab4 DNA for insertion into the similarly cut
pCOMB3H plasmid. The linearized DNA fragments were purified by a QIAprep™ gel
extraction kit and 250 ng of linearized pCOMB3H plasmid was incubated with 50 ng of
Fab4 insert with Gibco ligase overnight at room temperature. The ligase reaction was then heat-killed at 70ºC for 3 minutes.
Three electroporation experiments to obtain E. coli stocks of pCOMB-Fab4 transformed cells were performed. Twelve cultures were randomly selected for DNA
Miniprep, and ELISA testing. Twelve colonies were subcultured onto an LB/Carb (50
µg/ml) plate and into 3 ml aliquots of LB broth for overnight incubation at 37ºC. Their plasmid DNA was extracted using a QIAprep Spin Miniprep Kit. A small sample from each culture was held back for glycerol stocks (50% glycerol) and stored in the -80ºC freezer.
For IPTG induction of Fab4, the same 12 cultures were propagated in 20 ml of
defined medium broth plus 50 µg/ml carbenicillin per culture in 125 ml shaker flasks and
cultured for 9 hours at 37ºC and 250 rpms. They were cooled to 30ºC, induced with 1
mM IPTG and incubated overnight. Each culture was spun down for 15 minutes at 1500
x g, the cells separated from the supernatant and weighed. The cells from each culture
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were incubated on ice for 30 minutes with periplasm extraction buffer (0.2 mg/ml
lysozyme in 1.0 M NaCl, 1 mM EDTA, 5 µM PMSF, 50 mM tris·HCl, pH 8.0). They
were centrifuged for 30 minutes at 6000 x g, the pellet was discarded and the supernatant
diluted 10-fold and 100-fold and tested by ELISA for the presence of phagemid Fab4.
The supernatant from the same twelve cultures was also similarly diluted and directly tested for the presence of Fab4. The DNA samples were quantitated and restriction analyses were performed using Sac I/Xba I, Spe I/Nhe I, and Xho I/ Spe I endonuclease combinations. The clone expressing rFab and containing the appropriate insert was then repropagated in 100 ml of defined medium with 50 µg/ml carbenicillin. The DNA from the selected clone was sequenced using the four primers listed in Table 5.1.
Table 5.1: Sequence Primers Used for pCOMB-Fab4 PCR Amplification. Sequences of the oligomeric primers used to test the pCOMBFab4 sequence for the presence of the heavy and light chain sequences.
Primer Name Primer Sequence
pCBF4LC-F 5’-ATG CTT CCG GCT CGT ATG-3’
pCBF4LC-R 5’-AGG CCT TAC CAG CAC AGA CC-3’ pCBF4HC-F 5’-AAA TGG CGT CCT GAA CAG-3’ pCBF4HC-R 5’-AAT CAC CGG AAC CAG AGC-3’
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5.3.2 Library construction. Chemicals and Biological Reagents. Antibiotics
specific for isolating the host, [E. coli XL1-blue F+ pili, tetracycline, (Tet)], selecting for
the phagemid [pCOMB3H, carbenicillin (Carb)], helper phage [VCSM13, kanamycin],
isopropyl-β,D-thiogalactopyranoside (IPTG), ammonium sulfate, 3-(N-morpholino)
propane-sulfonic acid (MOPS) buffer, Tween-20, yeast extract and tryptone were
purchased from Fisher Scientific. Buffer and reagent chemicals were of the highest
analytical or molecular grade available from commercial vendors. The E. coli XL1-blue
strain, VCSM13 interference resistance helper phage, StrataPrepTM PCR Purification
Kits, PfuTurboTM polymerase and the MultiSiteTM Directed Mutagenesis Kit were
purchased from Stratagene (La Jolla, CA). Degenerate light chain and heavy chain
primers consisting of 65-base oligomer, 5' phosphorylated, LCCDR3RAN8, and a 68-
base oligomer, 5' phosphorylated, HCCDR3RAN9, were PAGE purified as per order
(SigmaGenosys). See Table 5.1 for primer design. The pCOMB3H phagemid was
received under a limited use agreement from Dr. Carlos Barbas, III, at Scripps Research
Institute in La Jolla, CA. High strength analytical grade agarose was purchased from
Bio-Rad. Super broth (SB) for culturing the phagemid infected E. coli XL1-blue strain
consisted of (30 g of tryptone, 20 g yeast extract and 10 g of MOPS) per liter and titrated to pH 7.0.
Some optimization for producing a library was necessary. The multi-site directed mutagenesis approach developed at Stratagene does not result in binary amplification per
cycle. Only one strand of the double-stranded circular DNA (dsDNA) is being copied per
cycle. We modified Stratagene’s procedure to improve results for the larger oligomers
needed to produce the naïve Fab Library. Nanogram amounts of primers were based on
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equimolar amounts of the forward primers per mutagenesis PCR reaction. We varied the
amount of PfuTurboTM in the QuikChangeTM Multi enzyme blend. See Table 5.2 for
PCR components. Optimized cycling parameters were 1 cycle at 95ºC, 30 cycles of 1min
at 95ºC, 1 minute at 63ºC and a final extension cycle of 4 minutes at 65ºC. All reaction
products were then DpnI digested to remove the template pCOMB-Fab4 methylated
DNA. The ssDNA was purified using 2 spin columns from a QIAQuickTM PCR
Purification kit and all DNA was pooled, then electroporated into 6 aliquots (1300 µl) of
electrocompetent XL1-blue strain E. coli cells utilizing a Bio-Rad E. coli Pulser and were
recovered in SOC medium by incubating for 1 hr at 37ºC. Aliquots were then plated in
three separate dilutions on Carb/LB agar plates, incubated overnight at 37ºC and colonies
counted the next day. The library cell culture’s DNA was subsequently Midi prepped
and the DNA was used for two rounds of phagemid amplification to produce a working
tertiary library for panning rounds
5.3.2 ELISA screen of phagemid rFab antibody fragments. Alkaline
phosphatase-ELISA for detecting phagemid rFabs utilized the following reagents and
solutions: 10 mM Na phosphate, 140 mM NaCl, pH 7.4 (EPBS); 2.5 mM Na borate, 100
mM boric acid, 75 mM NaCl buffer, pH 8.5, (EBS); rabbit anti-mouse IgG[F(ab')2]-
alkaline phosphatase (RAM-AP) (Pierce); phosphatase substrate tablets (Sigma);
diethanolamine and MgCl2 (Fisher Scientific). The ELISA substrate absorbance
measurements were made on a Labsystems Multiscan Ascent at 405 nm between 10 and
24 hours after addition of the substrate depending on the rate at which the individual plate’s response to the substrate was measurable. Fab4 protein standards were included on each plate.
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Table 5.2: PCR Parameters for the crFab Library Using the Multi-Site Directed
Mutagenesis Kit. The Pfu turboTM parameter was varied on the premise that it was a
part of the enzyme blend and increasing the starting DNA polymerase concentration may increase the end product provided the other enzyme(s) in the enzyme blend were not rate limiting.
Component Control MultiMix MultiMix enzyme
Reaction enzyme blend blend + Pfu turboTM
10x QuikChangeTM Multi reaction 2.5 µl 5.0 µl 5.0 µl
buffer
TM Sterile, Nanopure H2O 19.5 µl 35.0 µl 35.0 µl
dsDNA template pCOMBFab4 1.0 µl 2.0 µl 2.0 µl
Mutagenic primers:
LCCDR3RAN8 (144 ng/ l) 0 µl 2.0 µl 2.0 µl
HCCDR3RAN9 (151 ng/ l) 0 µl 2.0 µl 2.0 µl
dNTP mix 1.0 µl 2.0 µl 2.0 µl
Pfu turboTM 0 µl 0 µl 1.0 µl
QuikChangeTM Multi enzyme blend 1.0 µl 2.0 µl 1.0 µl
Total Reaction Volume 25.0 µl 50.0 µl 50.0 µl
25 colonies were selected at random and tested by ELISA for each protein for
panning rounds 2 and 3. A 2.5-fold color in excess of background was used as the cutoff
level.
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5.3.3 Panning procedure for phagemid Fab antibody fragments. The
procedure for selection of phagemid crFab antibody fragments followed the procedure of
Burton and Barbas with minor modifications. Panning of the selected proteins by was
carried out on Immulon 2 HB Flat Bottom MicrotiterTM Plates (Fisher Scientific) and all
of the suggested volumes were doubled accordingly. All solutions were either autoclaved or sterile-filtered for use with the panning cycles. Each pan cycle consisted of a recognition multi-step procedure followed by a replication multi-step procedure wherein the phagemid-Fabs were amplified.
The recognition step began with an overnight incubation of the desired target on an Immulon 2 plate at 4ºC, followed by a single wash step to remove the excess target, a blocking step with 1% BSA in tris buffered saline (150 mM NaCl, 50 mM tris·HCl, pH
7.5) with 0.5% (v/v) Tween 20 (BSA-TBST) at 37ºC for 1 hour which was also followed by its wash step. After this 100 l of phage stock was added to each well, beginning with the reamplified naïve library phage and then in subsequent cycles the phage stock obtained from the replication steps. The phage was incubated on the target protein for 2 hours at 37ºC. Removal of excess phage was then accomplished by filling the well with
BSA-TBST and pipetting vigorously up and down. After 5 minutes the TBST was removed. The stringency was increased with each cycle from one wash in the first recognition step to five washes in the second recognition step to ten washes in the third recognition step after which the phage were eluted with 100 µl of elution buffer containing 0.1 M HCl adjusted to pH 2.2 with glycine in the presence of 0.1% (w/v)
BSA. After incubation for 10 minutes at room temperature, the eluate was removed and neutralized with 6 µl of 2 M trizma base.
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Replication consisted of infecting 2 ml of freshly grown E. coli XL-1 blue strain
[O.D. 600 = 1 grown in with 10 µg/ml of tetracycline of super broth to select for the F+
strain] with 50 µl of the eluted phage. The 2 ml culture was incubated at room
temperature for 15 minutes. 10 ml the super broth medium with 20 µg/ml of carbenicillin and 10 µg/ml of tetracycline was pre-warmed to 37ºC and then added to the phage- infected 2ml culture and incubated for 1 hour at 37ºC on a shaker at 300 rpms. The total concentration of carbenicillin was increased to 50 µg/ml and incubation continued at
37ºC for an additional hour on a shaker at 300 rpms. At this point phage particles were rescued by the addition of VCSM13 (1012 plaque forming units) interference resistance
helper phage in the phagemid libraries and transferred to 100 ml of super broth with the
same antibiotics and incubated on the shaker for 2 hours. Kanamycin was added to the super broth at a final concentration of 70 µg/ml, the temperature was dropped to 28ºC and culturing continued overnight. The cultures were spun down in a Beckman Avanti J-25 I centrifuge 3000 x G (JA10 rotor) for 20 minutes at 4ºC. The phage stocks were then precipitated out of the medium for several hours over ice with ice cold 4% PEG 8000/
0.4M NaCl (final concentration in the medium solution). The precipitate was then spun down at 15,000 x G (JA10 rotor) for 20 minutes at 4ºC and the supernatant discarded
(Biohazard level 2). The pellet was re-suspended in 1 ml TBST and centrifuged for 5 minutes at 14,000 rpms in an Eppendorf microcentrifuge 5415C and stored at 4ºC.
50 − 100 µl of phage stock was used to infect 2 ml of E. coli grown with Tet selection to an O.D. 600nm of 1.0. After a 15-min. incubation at room temperature, 10 ml of super broth-(10 µg/ml Tet plus 20 µg/ml Carb) broth was added and incubated at
37°C for 1 hr. 100 µl from this starting culture volume was either directly plated or used
98
as the starting dilution volume spread on 100 µg/ml Carb-LB agar plates. A minimum of three 10-fold dilutions per panning cycle were incubated overnight at 37°C and colonies were counted the next day. The aforementioned times were critical to the success of the panning experiment.
5.4 Results
5.4.1 Selection of the pCOMB-Fab4 clone. Three electroporation experiments to obtain E. coli stocks produced only a few colonies but for this experiment it was not the quantity but the quality of the colonies that was important. Only one of the electroporation experiments worked even though all three had time constant values in the acceptable range of 4.5 to 5.0. See Table 5.3 .below.
Table 5.3: Electroporation Results for pCOMB-Fab4 Transformation in E. coli. See
Appendix B for a description of the electroporation procedure.
Electroporation Electroporation 200 µl colony 20 µl colony 2 µl colony
Experiment Time Constant 1 count count count
1 5.0 msec 2 23 0
2 5.0 msec 0 0 0
3 5.0 msec 0 0 0
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Table 5.4: DNA Results for the 12 pCOMB-Fab4 Transformed Colonies.
Spectrophotometric readings were taken directly using the DNA program on the spectrophotometer and 20x dilutions of each DNA sample.
Colony # 260 nm 280 nm 320 nm Concentration 260nm/280nm
µg/µl ratio µg/ml
1 0.087 0.054 0.009 3.9 1.6
2 0.077 0.047 0.004 3.6 1.6
3 0.094 0.062 0.017 3.8 1.5
4 0.103 0.063 0.010 4.7 1.6
5 0.083 0.053 0.009 3.7 1.6
6 0.145 0.087 0.011 6.7 1.7
7 0.092 0.060 0.014 3.9 1.5
8 0.080 0.051 0.009 3.5 1.6
9 0.087 0.056 0.009 3.9 1.6
10 0.085 0.055 0.011 3.7 1.5
11 0.084 0.052 0.009 3.8 1.6
12 0.082 0.054 0.006 3.8 1.5
The quantitative DNA results for the twelve colonies are given in Table 5.4. When a 20x dilution of the DNA is used the concentration of the DNA is read directly from the UV
260nm reading. The readings are automatically sequentially read in the DNA program on
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the spectrophotometer and the concentration is calculated in µg/ml by the DNA program.
The quality of the DNA is measured by the 260/280 ratio. The closer the ratio is to 2.0
the higher the quality of the DNA. This is dependent largely on the thoroughness of the
first lysis step. Clumps of cells do not lyse well and contaminating proteins will lower
the quality of the DNA.
Figure 5.1 graphs four parameters that were measured for each culture, two with
respect to growth of the cultures and the second two with respect to the rFab productivity
of the cultures. The fastest growing culture, number 5, was not the most productive one.
The productivity of culture number 9 made it the obvious choice for taking this clone to
the next level and. The Midi prep of this culture produced very high quality DNA at a
concentration of 1.234 µg/µl (260 nm/280 nm ratio = 1.9).
5.4.2 Library construction. Figure 5.1 is a schematic diagram of the pCOMB-
Fab4 phagemid. The engineered SacI and Spe I endonuclease restriction sites in both the
pET28b plasmid and pCOMB3H phagemid allowed for single-step transfer of our fourth
recombinant crFab mutant from the pET28b plasmid into the pCOMB3H phagemid
plasmid. The locations of the CDR loops are indicated by asterisks in the figure with the library primer sequences indicated above the appropriate CDR loop. Only the forward primers, LCCDR3RAN8 and HCCDR3RAN9 shown for each chain were used as per
instructions for the QuikChangeTM Multi Site-Directed Mutagenesis Kit in the crFab
Library PCR reaction. Their method is based on synthesis of single-stranded circular
DNA (ssDNA). With G and T in the third position, premature stop codons are minimized to one out of a possible three.
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Comparison of 12 Cultures
6.0
5.0 O.D. 600 Cell Weights 4.0 in Grams
3.0 O.D. 405
2.0 Periplasm ELISA Signal
1.0 Supernatant ELISA Signal O.D. 405 ELISA Signal O.D. 405 ELISA 0.0
Grams of Cells Versus O.D. 600 O.D. Versus Cells Grams of 1921011123456 7 8 -1.0 Culture #
Figure 5.1: Relative ELISA Response of 12 pCOMB-Fab4 Clones. The growth of
the cells was estimated by O.D. 600 in solution and wet cell weights were determined
after the cells were spun down. The ELISA was performed in the usual manner and
the O.D. 405 nm response to alkaline phosphatase utilization of the pNPP substrate
was measured on the Labsystems Multiscan Ascent instrument. The background was
subtracted from each ELISA response.
.
Starting with pCOMB-Fab4 as the forerunner for our library, the DNA sequence was confirmed by PCR sequence analysis. The pCOMB-Fab4 sequence has been
102
Light chain CDR3
5’-CT GCA ATG TAT TTC TGT CAG NNK NNK NNK NNK-NNK NNK NNK-NNK TTC GGT GCT GGG ACC AAG GTG-3’
Heavy chain CDR3
5’-GTC TAT TAC TGT GCA AGA TGG NNK NNK NNK NNK NNK NNK NNK NNK NNK TA
TGG GGC CAA GGC ACC AC-3’
SacI XbaI XhoI SpeI ATG ATG
* * * TAA * * *
A fabL RIBS PelB fab RIBSOmp H NheI g II Z I lac A TA
ColE1 ori
Ampr
pCOMB-Fab4
Figure 5.2: pCOMB Phagemid Vector Containing the Light (fabL) and Heavy
(fabH) Sequence Data. * Location of mutatable sites (Complementarity Determining
Region loops) with the degenerate DNA primer sequences for the library shown. In coding for degenerate primers the N was A, T, C or G. K was G or T. Limiting the final base to G or T minimized premature STOP codons to one out of a possible three while permitting encoding of all 20 amino acids. RIBS, ribosome binding site. OmpA and PelB are leader sequences targeting the periplasm. Ampr is the ampicillin
(carbenicillin) resistance gene. ATG is the start codon and TAA is the stop codon.
LacZ is the IPTG-inducible lactose response gene. ColE1 ori is the origin of replication site.
103
deposited with GenBank Data Libraries, acquisition number AY254174 and the data are
also recorded in Appendix D.
As shown in Table 5.5, a preliminary study was done to test the transformation
efficiency of the ssDNA library in electrocompetent E. coli XL1-blue cells. In the preliminary PCR test reactions, varying the PfuTurboTM in the QuikChangeTM Multi
enzyme blend produced greater than a 5-fold higher number of transformants per reaction
(31 transformants x 2 as opposed to 100+ transformants and 219 transformants). Since
both PfuTurboTM and the QuikChangeTM Multi enzyme blend are Stratagene products we
assumed that PfuTurboTM was in the enzyme mix. This did not confirm our supposition
that PfuTurboTM was actually in the enzyme blend, but it did confirm that increasing the
amount of starting polymerase molecules could increase the amount of end product
(number of transformants).
A preliminary test of multi-site mutagenesis with 1 µl of the purified ssDNA in
1.0 ml of SOC medium produced 100+ colonies when 2 µl of the transformed bacteria were plated in one test and when repeated a second time produced 219 colonies per 2 µl
SOC medium. The calculations of transformation efficiency were as follows.
1. Transformants for the Kit mix:
a. 1 µl DNA x 45 (diluted into 45 µl cells) x (1045/45 SOC dilution factor) x
1000 µl/ml x (31 colonies/2 µl) = 1.62 x 107 transformants/ml.
b. Average transformants: [(1.62 x 107 transformants) *2]/2 = 1.62 x 107.
2. Transformants for the Kit mix plus PfuTurbo™:
a. 1 µl DNA x 45 x (1045/45) x 1000 µl/ml x (100 colonies/2 µl) x 1 ml =
5.23 x 107 transformants.
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Table 5.5: Colony Count Results from Preliminary PCR Transformations. Each
PCR reaction’s ssDNA was transformed into 40 µl of E. coli XL1-blue cells. A sheet was confluent colonies so close together that individual colonies could not be counted. A lawn was a less dense distribution of colonies than a sheet but still might have areas on the plate where the colonies grew together and could not be accurately numbered. The
Dpn I treatment to remove the parent methylated DNA and those results are marked in bold.
200 µl colony count 20 µl colony count 2 µl colony count
Experiment or Before After Before After Before After
control Dpn I Dpn I Dpn I Dpn I Dpn I Dpn I
treatment treatment treatment treatment treatment treatment
No primers + sheet 200-300 lawn 7 >400 0
kit mix
Primers + kit sheet lawn lawn 200-300 400-500 31
mix #1
Primers + kit sheet lawn lawn 200-300 200-300 31
mix #2
Primers + Pfu sheet sheet lawn lawn 400-500 100+
I + kit mix
Primers + Pfu N/A sheet N/A lawn N/A 219
I + kit mix
pUC18 control N/A lawn N/A 154 blue/ N/A 16 blue/
311 total 41 total
105
b. 1 µl DNA x 45 x (1045/45) x 1000 µl/ml x (219 colonies/2 µl) x 1 ml =
11.44 x 107 transformants.
c. Average transformants: (52,250,000 + 114,427,500)/2 = 83,338,750 =
8.33 x 107 transformants
A 20-fold dilution of the ssDNA was measured on the spectrophotometer and the
yield of DNA calculated to be 5.0 µg. The entire sample of 5.0 µg of ssDNA was divided
into 6 aliquots and transformed into a total of 1300 µl of electrocompetent cells. The six
transformation reactions were combined in a volume of 23 ml of SOC and incubated for
1 hour at 37ºC. Spreading 100 µl of this culture on LB/Carb plates produced a sheet for the undiluted culture, the equivalent of 10 µl produced a lawn, the equivalent of 1 µl was countable at 482 colonies and the equivalent of a 0.1 µl spread on an LB/Carb plate produced 52 colonies. 23 ml x 482 transformants/µl x 1000 µl/ml = 11,086,000 transformants. 23 ml x 520 transformants/µl x 1000 µl/ml = 11,960,000 transformants.
Averaging the two figures for the two colony counts, the result of the optimization strategy was a nominal diversity of 1.16 x 107 colony forming units. The pUC19 three-
site mutagenesis control supplied with the kit produced 154 blue out 311 colonies for a
triple mutant transformation efficiency of 50%.
5.4.3 Selection of phagemid antigen-binding Fab antibodies. The primary
library was amplified to 1.6 x 108 colony forming units per ml and then this was further amplified to 3.3 x 109 colony forming units per ml for a tertiary library. This was done to
assure that adequate diversity of the library would be represented in each subsequent
panning.
106
Six to seven panning rounds were attempted for each protein but the results in general were poor after the third panning round for lysozyme, BHMT, CBS and CGL and after the first panning round for FlgR, MerR and p24. The unsuccessful panning rounds were actually started on the same day. Therefore the results reflect the conditions of the culture on that date and not on the proteins being panned. Panning for FlgR, MerR and p24 proteins had to be repeated. In Figure 5.2 the Panning Results are offset by two rounds because those three proteins’ panning cycles were started after the first four had already undergone two panning cycles. The panning results bottomed out on all seven cultures on the same day. FlgR, HIV capsid p24 and MerR panning rounds had to be repeated and those panning results are recorded in Table 5.6.
Table 5.6 showed that we obtained amplification of our selected rFabs against the given seven antigen proteins after three panning cycles. Since the parent rFab was derived from murine Mab25.3 specific for HIV I capsid protein p24, p24 was included as a control (Kovari et al., 1995, Momany et al., 1996). Notably, it was neither better nor worse than the other six protein targets in the number of transformants per panning round.
On the other hand, HIV I capsid protein p24 did surpass the other protein targets in the percentage of transformants that are carrying phagemid-rFabs specific for the target, but only ranges from 2- to 5-fold greater when compared with the other phagemid- rFabs (Table 5.7). The effective total number of phagemid-rFabs is derived from the total number of colony forming units times the percentage of positive ELISA responses. The effective total number of phagemid-rFabs produced against their target at the end of the third panning cycle has four of the six other protein targets outperforming the HIV I
107
Panning Results for Lysozyme, BHMT, CBS and CGL
1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06 Lysozyme BHMT 1.00E+05 CBS 1.00E+04 CGL
1.00E+03
1.00E+02
1.00E+01
1.00E+00 1234567
Panning Results for FlgR, HIV capsid p24 and MerR
1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06 FlgR 1.00E+05 HIV capsid p24 Mer R 1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00 123456
Figure 5.3: Panning Cycle Results for the Seven Proteins. See text for details.
108
capsid protein p24 in total numbers of target specific phagemid-rFabs produced at the end of that cycle.
In selecting a sub library to a specific protein, the first cycle of panning produces minimal selectivity to that protein. A single wash cycle was employed which removed only the unbound crFabs. In the second cycle of panning the specificity to the target was enhanced by increasing the stringency to five washes as well as amplification of the total library size, but now a small sampling of 25 transformed colonies produced detectable
ELISA results to the target in four out of seven of the selected targets. By the third panning cycle the stringency was further increased from 5 to 10 washes causing a reduction in total library size back to levels seen in Pan Cycle 1. The amplification step, however, had produced a significant increase in ELISA response to transformed colonies.
The effective number of crFab phagemids, based on the percentage of ELISA-positive times the total number of clones per crFab library, is lower than the total number of crFab phagemids for three reasons. Our cut-off level of 2.5-fold above background would eliminate weakly positive target crFab phagemids. The procedure for obtaining crFab phagemids cannot exclude plastic-binding phagemid crFabs. Finally, “insert-less virions” may have been propagated as well (Soltes et al., 2003). In Figure 5.4 panel a pan cycles 3 through 5 were analyzed. Because these represented pan cycles higher than 3, it was suspected that propagation conditions for the libraries had suffered from adverse overnight culture conditions after pan cycle 3 and those conditions decreased the library sizes drastically without affecting the viability of those E. coli cells containing insert-less virions.
109
Table 5.6: Colony Count Results for Library Panning: Abbreviations used for target proteins were Hen egg white lysozyme,
HEWL; human betaine homocysteine methyl transferase, BHMT; human cystathionine β-synthase, CBS; human cystathionine γ- lyase, CGL; bacterial FlgR; bacterial transposon21 MerR; and HIV I capsid protein, p24.
Pan Cycle HEWL BHMT CBS CGL FlgR MerR p24
1 2.10 (0.47) 1.67 (0.83) 1.63 (0.41) 3.17 (0.43) 4.10 (0.74) 3.89 (0.05 4.00 (0.08)
2 518. (208) 417 (98) 843. (224) 1005 (368) 1970 (89) 1110 (57) 1450 (83)
3 107 (62) 87.4 (32.0) 41.9 (18.3) 143 (84) 0.71 (0.48) 53.9 (1.3) 8.62 (4.74)
Each number is the Average of three colony counts with all values x106. Numbers in parentheses represent the variance between colony counts within the serial dilutions of each of seven protein targets.
110
Table 5.7: ELISA Results for Library Panning of Seven Proteins: ELISA Response for 25 Cultures per protein target. A signal to noise ratio > 2.5 was used as the cutoff for positive response. Target proteins were the following: Hen egg white lysozyme, HEWL;
human betaine homocysteine methyl transferase, BHMT; human cystathionine β-synthase, CBS; human cystathionine γ-lyase, CGL;
bacterial FlgR; bacterial transposon21 MerR; and HIV I capsid protein p24, p24. The bacterial flagellum regulatory protein FlgR
remained at a stationary percentage.
Pan Cycle HEWL BHMT CBS CGL FlgR MerR p24
2 (0/25) (4/25) (0/25) (0/25) (12/25) (5/25) (3/25)
0% 16% 0% 0% 48% 20% 12%
3 (6/25) (8/25) (3/25) (8/25) (12/25) (7/25) (16/25)
64% 24% 32% 12% 32% 48% 28%
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The pCOMB-rFab-gIII phagemids were more slowly detected by ELISA utilizing rabbit anti-mouse alkaline-phosphatase linked secondary antibody (RAM-AP) than the original rFab4 protein expressed in the pET28 plasmid system. Since it was a surface-to- surface interaction between the CDR loops of the rFab and its cognate target protein, the rFab alone was considerably smaller than the pCOMB-rFab-gIII phagemid. The secondary antibody was hindered but not prevented from interacting with this large complex.
The unexpected result from the panning of the seven protein targets was that HIV
I capsid protein p24 did not produce a significantly better sub library toward its target than the others by the measurement of ELISA responses of the 25 selected colonies multiplied by the total numbers of colonies. One may speculate that follow-up experiments to characterize the anti-p24 rFabs from these phagemids may reveal unexpected results as well. Based on the information supplied with the Stratagene kit, it was expected that ~10% of the library would remain as unmodified rFab4.
In Figure 5.4 the DNA endonuclease cut pattern for the library is evidenced by a similar cutting pattern to the pCOMB-Fab4 template. Although differences in cutting pattern can be expected within a library due to the random introduction of new endonuclease restriction sites and secondary structure, the majority of mutated DNA did not lose our mutant’s pattern of endonuclease restriction in the multi-site single-step process of developing it. The results from agarose gel electrophoreses demonstrated that specific single-clone DNA within the library for each protein pan did have endonuclease restriction cutting patterns similar to the parent DNA from which the library was derived.
Two representative DNA endonuclease restriction patterns of specific clones that gave a
112
A.
pCOMB - pCOMB- pCOMB - pCOMB- pCOMB - Fab4 p24 p24 p24 1o Lib 3rd Pan 4th Pan 5th Pan R XhSp XhSp SaXb SpNh SpNh EcoR XhSp SpNh SpNh EcoR EcoR SaXb SaXb XhSp SaXb SpNh XhSp Eco EcoR SaXb Uncut Uncut Uncut Uncut 100bp Markers Markers 100bp Uncut 100bp Markers Markers 100bp 1 Kb Markers 1 Kb 1 Kb Markers 1 Kb
10,000
2000 1500
750 500
B.
pCOMB pCOMB pCOMB Fab4 FlgR p24 R SaXb SpXh Uncut 10,000 SpNh SaXb SpXh SaXb SpXh SpNh EcoR Uncut Uncut EcoR Eco SpNh/ 1Markers Kb 100bp Markers Markers 100bp 100bp Markers Markers 100bp 1 KbMarkers
2000 1500
1000 750
500
250
Figure 5.4: Agarose Gels of Restriction Endonuclease Cuts. Panel A, endonuclease restriction cuts of the pCOMB-p24-gIII subset of the library DNA from three panning cycles of HIV I capsid p24. The pCOMB-1oLib is part of the same gelexcept that it has been photo enhanced for clarity. Panel B, endonuclease cuts of two representative subsets of library DNA,pCOMB-FlgR and pCOMB-p24. SaXb = SacI/Xba I, XhSp =
XhoI/SpeI, SpNh = SpeI/NheI, and EcoR = EcoRI are endonuclease restriction cuts.
113
positive ELISA signal to p24 and FlgR are shown in Figure 5.4b. The original pCOMB-
Fab4 was included as a positive control.
5.5 Discussion
The pCOMB3H plasmid was chosen because it is suitable for the expression of
both the light and the heavy chains in a single phagemid utilizing the E. coli XL-1blue
strain of bacteria under the influence of signal peptides (Burton & Barbas, 1993, and
Burton, 1995). Super infection of E. coli with more than one pCOMB3H phagemid (or phage) per cell does not occur. Thus, each pCOMB-infected colony represents a single
Fab clone. This plasmid has a copy of gene III and a phage packaging signal sequence.
Supplying the missing factors for producing the infective phage particles with VCSM13
interference resistance helper phage minimizes production of insert-less virions, a
common problem when the phage is used. We wanted to avoid the high avidity of low
affinity Fabs that a gene V insertion in the major coat protein would produce. The
VCSM13 interference resistance helper phage supplies the missing factors to export
infective phage particles to the medium.
The nominal diversity of 1.16 x 107 obtained with this technology is significant.
The library was expressed in the XL1-blue strain of E. coli that had demonstrated a
transformation efficiency of 4 x 1010 with a pUC19 control. A lower efficiency of
transformation was expected for the ssDNA library than for a dsDNA library. However,
library sizes of 106 to 109 are usual for Fab libraries and 1010 possible for dsDNA scFv
libraries (Lou et al.). Therefore, our crFab library is within the expected range, although
on the low end. Increasing the overall size and diversity of the library could have been
114
accomplished by increasing the number of transformation reactions with the same amount of DNA and with additional PCR amplifications utilizing the kit with the
remainder of the primer stocks to obtain more ssDNA for transformation.
Seven proteins available in the laboratory (hen egg white lysozyme, HEWL;
human betaine homocysteine methyl transferase, BHMT; human cystathionine β-
synthase, CBS; human cystathionine γ-lyase, CGL; bacterial FlgR; bacterial transposon21
MerR; and HIV I capsid protein, p24) were selected for preliminary evaluation of this
phagemid crFab library for the ability to recognize diverse targets. To demonstrate that
the target rFab sub libraries generated are also to each target useful for co-crystallization
requires us to generate a milligram supply of each rFab to its specific target and begin co-
crystallization trials. Two strategies are (1) to re-clone each crFab into the pET28b
plasmid or (2) to remove the gene III product from the pCOMB-rFab for expression and
purification of the target-specific crFab at high levels.
Although phage libraries are purported to give better overall results than phagemid libraries with regards to finding antibodies quickly to a target (Lou et al.), for
our purposes this phagemid library is preferable. Our goal is to create high-affinity
crFabs for co-crystallization with novel protein targets in which the physical properties of
the co-crystallization Fab are unchanged. High affinity antibodies imply stronger
interactions between the target and its cognate crFab.
115
CHAPTER 6
CONCLUSIONS AND FUTURE DIRECTIONS
6.1 Conclusions
Recombinant antibody fragment biotechnology in this fast-paced age of technology is continually developing with older techniques being modified or completely revamped. Chapter 2 focused on the many applications to which antibody fragment
biotechnology is currently being developed with an eye to the possible future applications. Since the advent of phage display and other enrichment technologies and the more recent application of this technology to display antibody fragments, new uses for recombinant antibody fragments are emerging rapidly. A recombinant antibody fragment may have either research or commercial value as a tool for probing difficult areas of biology or helping to bring about a cure for disease processes. The general
usefulness of these molecules, relative stability under adverse conditions, and well- known structural characteristics and genetics make them attractive biotechnological development candidates. Current applications are coming rapidly into broader usage and future developments are exciting to explore.
In Chapter 3, the Fab4 mutant was compared with other earlier mutants and a subsequent mutant for stability, ease of production and crystallizability. We have expressed five mutants of a mouse recombinant antibody fragment (Mab25.3) whose
116
epitope was HIV I p24 capsid protein to the periplasmic space of Escherichia coli and
into the culture medium. The mutants are His-tagged to aid in purification. The rFabs
were detected in both the periplasm and the defined medium, and exported at high levels
from Escherichia coli strains BL21(DE3) and BL21(DE3)-RIL to the defined medium.
A rapid purification protocol was developed for extraction of pET28-Fabs from the
medium. Crystallization of the mutants was the main deciding factor in the selection of
the rFab4 mutant as the basis for building a library for crystallizing proteins. rFab4 was
selected over the other mutants based primarily on its high level production and rapid
formation of small crystals. The other rFabs either did not crystallize or did so at such slow rates as to make them unfeasible as co-crystallization reagents. A summary of this work will be submitted to the journal of Protein Expression & Purification.
In Chapter 4, a preliminary report was given on efficient production of Fab4 in a bioreactor process. Whereas this holds promise for improving our yields under certain specific conditions of growth for Fab4, and may be easily extended to crFabs, there are a
number of conditions that have not yet been optimized and need further study. A
preliminary study indicated that at least 10 mg/L might be achieved.
In Chapter 5, it was demonstrated that there were some reasonable results from
our preliminary studies on the co-crystallization library that make this Co-Crystallization
Antibody Fragment Library project look hopeful for taking it to proof of concept. A non-
immune phagemid recombinant antibody fragment (Fab) library was generated to
facilitate crystallization of proteins that have not previously been crystallized. A nominal
diversity of 1.06 x 107 was obtained with a polymerase chain reaction utilizing multi-site
directed mutagenesis technology. The PCR reaction was rapid and effective. These
117
results have led to a proposal to further utilize this library as a co-crystallization strategy
and the proposal has been submitted to the governing authorities. The data that are
relevant to the technique of obtaining a large library by multi-site directed mutagenesis
will be submitted for publication to the journal of BioTechniques.
6.2 Future Production and Development of the crFab Co-Crystallization Library
Optimization of production of crFabs in a bioreactor process is the next step for
scale-up of product. Our goals are to determine the optimal concentration of IPTG and
the optimal temperature to begin IPTG induction in shake flask culture. We would also like to know whether Mg2+ concentration affects Fab4, and hence the Fab library,
production.
Once we have determined the optimal magnesium and IPTG concentrations and
the best temperature for induction then bioreactor experiments could be conducted with
the following goals in mind: (1) determine whether glutamate and amino acids affect
Fab4 production and (2) determine whether NH4OH or NaOH is a better base.
Once the bioreactor process has been optimized for rFab4 then it follows that the
crFabs could easily be produced under the same conditions. The final step to taking this
project to proof of concept is to do a scale up of crFab production from each positive
result from the ELISA screening against each protein target. It has not yet been
determined whether or not to keep the crFab in the pCOMB3H phagemid plasmid or to
transfer it to the pET28b plasmid as the most efficient means of producing it in E. coli.
Switching it back into another strain of E. coli may be more productive than the XL1-
blue strain permits.
118
High throughput means a rapid turnover of a large number of items (chemicals tested, targets panned, proteins crystallized, etc.) and is essential for dealing with the large numbers of unknown protein structures to which this technology could be applied.
It can be envisioned that a high-throughput co-crystallization of crFabs plus their targets could follow the production of a high throughput panning process.
Robotics would be essential at this stage to bring high throughput to pass. The
human factors of fatigue and confusion when too many repetitions of the same thing are
done in a short period of time, and the physical injuries those repetitions cause, make it essential to have a robot(s). The robot that is presently available did not handle the
ELISA process well. The Biomek 2000 robot was not rugged enough to handle numerous incubation times required for a complete ELISA. It handled shorter programs well but could not carry through with a complete ELISA. It locked up at the critical final step of the process. Robots need to be designed to carry out this research to completion including handling the cultures.
Also a larger fermentation facility is needed to carry on panning of such large numbers. Each panning involves a 30 to 50 ml culture per protein panned. This also needs some form of automation and possible fermenter rooms that have the temperature of one room maintained at 37ºC and another maintained between (25ºC -30ºC) with some sort of shuttle mechanism to move the cultures between the two temperature-controlled areas. When these problems have been addressed there will probably be a stream of
results to look forward to obtaining. Hundreds, if not thousands, of new structures could
soon be coming off the high-throughput co-crystallization “assembly line.”
119
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139
APPENDIX A
LIST OF ABBREVIATIONS
2-D or 3-D NMR two-dimensional or three-dimensional nuclear magnetic resonance
Ab-AP alkaline-phosphatase coupled antibody
BRET bioluminescence resonance energy transfer
BSA bovine serum albumin
CDR complementarity determining region (hypervariable region)
CL constant domain of the Fab light chain
CH constant domain of the Fab heavy chain
DIP delayed infectivity panning dsFv disulfide-stabilized Fv fragment
ELISA enzyme-linked immunosorbent assay
EYFP enhanced yellow fluorescent protein
Fab antibody fragment having both a variable domain and a constant
domain
Fc constant domain tail fragment
Fv smallest heterodimer made up of antibody VH and VL domains
containing all the necessary information to interact with its specific
antigen
140
HER2 human epithelial growth factor receptor 2
HIV human immunodeficiency virus
HuCAL fully synthetic human combinatorial antibody libraries
IgA immunoglobulin A, (6 α chains) found in the intestinal mucosa
IgD immunoglobulin D, (2 δ chains), first antibody formed in humans
(major mouse immunoglobulin)
IgE immunoglobulin E, (2 ε chains), antibody associated with
anaphylactic shock IgG “gamma globulin,” (2 γ chains) smallest circulating immune antibody
IgM “macro globulin,” (10 µ chains) largest circulating antibody, existing
as a pentamer
IPTG isopropyl-β-D-thiogalactopyranoside, the lactose analog used to
induce the bacterial T7 promoter to start protein production
Mab monoclonal antibody
MGIF Molecular Genetics Instrumentation Facilities, University of Georgia,
Athens, GA
OS-BLIA open sandwich bioluminescent immunoassay
PDB Protein Data Bank
ScFv single-chain variable domain fragment
SIP selectively-infective phage
TM trade mark
VH heavy chain variable domain
VL light chain variable domain
141
APPENDIX B
SHORT PROTOCOLS
Preparation of defined medium and super broth
For protein production in crFab transformed E. coli, the following materials were added together. Sufficient de-ionized water was autoclaved so that the final volume was
1.00 L. The carbon source of 40% (w/v) glucose was added to attain 4.0 g /L final concentration in the start medium. Amino acid mixes, vitamins, antibiotics and ferrous sulfate solutions were prepared separately in advance and filter sterilized before final addition to the medium. M9 (2.0 g NH4Cl, 6.0 g KH2PO4, 13.6 g Na2HPO4 anhydrous or
25.6 g Na2HPO4 heptahydrate per liter of final medium) was prepared as a 10-fold
concentrated buffer solution in advance and autoclaved. Amino acid mix I consisted of
all L-amino acids except methionine, tryptophan, tyrosine and phenylalanine each at a concentration of 4 mg/mL and stirred for 1 hour to fully dissolve all of the amino acids.
Amino acid mix II consisted of L-tryptophan, L-tyrosine and L-phenylalanine adjusted to pH 8 with NaOH to improve solubility of these amino acids. Precipitated excess amino acids were removed via sterile filtration. The amino acid L-methionine solution was prepared separately at a concentration of 20 mg/mL. Final amount of each amino acid is
40 mg/L or at saturation. The following filter-sterilized vitamins were added to the medium to a final concentration of 1 mg/L per vitamin: riboflavin, niacinamide, pyridoxine monohydrochloride and thiamine. Additional salts required for growth were
142
0.02 M MgSO4 and 25 mg/L (90 µM) FeSO4 x 7H2O. Antibiotics required for the two
host strains were the following: 30 mg/L kanamycin for the (DE3) and RIL strains and
an additional 34 mg/L chloramphenicol for the RIL strain.
Super broth for culturing E. coli consisted of (30 g of tryptone, 20 g yeast extract
and 10 g of MOPS) per liter and titrated to pH 7.0.
Electroporation E. coli Transformation Protocol. DNA from the various
mutants was transformed into E. coli BL21(DE3) cultures and E. coli BL21(DE3)-RIL
cultures using the following protocol. Advance preparations required that appropriate
antibiotics be added to LB agar plates and enough of these plates had to be poured to
allow 3 plates per transformation reaction. Kanamycin (30 µg/ml) plates for isolating
BL21(DE3) strain of E. coli and 30 µg/ml kanamycin with 34 µg/ml chloramphenicol
plates for isolating BL21(DE3)-RIL strain of E. coli. In advance these plates were warmed to 37oC in the Fisher Scientific Isotemp Incubator
An E. coli Pulser TM (Bio-Rad) was used for electroporation transformation of E. coli host cells. 1.0 ml aliquots of SOC media were prewarmed to 37oC in the water bath
for each DNA transformation reaction to be done. The leads were put into the “TO
SHOCKING CHAMBER” sockets and the Pulser was set at 2.50 volts maximum for 2
mm gap cuvettes or 1.8 volts for 1 mm gap cuvettes. The E. coli Pulser cuvette holder and cuvettes were prechilled on ice. 100 µl of the appropriate electrocompetent
[BL21(DE3) or BL21(DE3)-RIL)] E. coli cells were removed from the -80oC freezer,
transferred to an Eppendorf tube and placed on ice. 1µl of the appropriate DNA (either
undiluted or diluted 1:100) was mixed gently with a pipette tip to avoid bubbles and
143
incubated on ice for 60 seconds. The cells were transferred with an Eppendorf pipette tip to a prechilled cuvette avoiding bubbles. The cuvette had to be snapped sharply to force the cells to the bottom of the cuvette. The cuvette slid into the cuvette holder by a one-
way design and both of the PULSE buttons were held down at the same time. If it
sparked, the DNA was too concentrated or the cuvette was too wet. The “TIME
CONSTANT” was checked for each sample by holding down both “SET VOLTS” and
“ACTUAL VOLTS” buttons at the same time. 4.5 to 5.0 was the acceptable range and the number was recorded for each sample
The electroporated cells were transferred to 37oC SOC media aliquot utilizing the
sterile bulb pipette that comes with the cuvette. The culture tubes were shaken at 37oC
and 225-250 rpms for 60 minutes on the New Brunswick Classic Series C24 Incubator
Shaker. 500 µl, 50 µl and 5 µl of the selected culture were pipetted onto separate
antibiotic plates (3 plates per transformation.) Plates were dated and labeled
appropriately. Cells were spread to form a bacterial lawn. 150 µl and 195 µl, respectively, of SOC media was added to the smaller volumes of cells to help spread them and incubated overnight in the Fisher Scientific Isotemp Incubator at 37oC. Plates
were checked for transformants the next day. Appropriate colonies were picked for stock
cultures and culture in 1 ml of LB at 37oC overnight. 50% (vol/vol) glycerol was added
to each stock culture, labeled and stored in cryotubes in the -80oC SO-LOW Ultra-Low
Freezer (Hanahan, 1983, Dower et al., 1985)
ELISA procedure. Purified monoclonal (HIVCA FS II at 30 mg/ml in TE
buffer) or the recombinant HIV I capsid p24-6His (2 mg/ml) was diluted to 5 µg/ml with
144
ELISA diluent borate saline buffer, pH 8.0. 0.1 ml of the diluted p24 protein was added to each of the test wells in a 96-well Immulon flat-bottom EIA/RIA plate. For each test well there was a corresponding control well that contained 0.1 ml of borate saline, pH
8.0. The plate was then covered with parafilm or tape and incubated overnight at 4ºC.
After being removed from the refrigerator, the immunogen was shaken into the sink and blotted. The plate was washed one time with wash buffer (0.05% Tween 20, 10 mM tris·HCl, pH 7.5).
The wells were filled with blocking solution (1% (w/v) BSA in EPBS) and allowed to incubate in the wells for a minimum of 2 hours at room temperature. The plate was again washed one time with wash buffer as above.
The 0.1 ml samples and/or their dilutions and Fab controls were then applied to the wells and allowed to stand at room temperature for 2 hours. The plate was again washed 3 times with wash buffer.
The GAM-AP or RAM-AP (secondary antibody) was diluted 1:500 and 0.1 ml was applied to each well. The secondary antibody was allowed to incubate for 2 hours at room temperature. The plates were washed 3 times with wash buffer and 0.2 ml of 0.6 mg/ml p-nitrophenylphosphate substrate in 2 mM MgCl2, 1.0 M diethanolamine, pH 9.6 buffer was then applied to each well in the plate. The yellow color of the reaction product was followed, usually taking about 30-60 minutes to develop, using a 405 nm filter in a 96-well scanner utilizing the Ascent software on a Labsystems Multiscan
Ascent and the data recorded. Analyses of the data were made using the Excel program software.
145
Bacterial culturing and centrifugation. Shake cultures were maintained at
appropriate temperatures in a Classic Series C24 Incubator Shaker TM (New Brunswick
Scientific). Aeration was achieved at 225-300 rpms in either Fernbach, Erlenmeyer or
baffled flasks having at least a 5 to 1 ratio of flask volume to culture volume.
After the maximum allotted time for culturing was past, the bacteria were separated from the liquid media via an ultra centrifuge, an Avanti J-25 I CentrifugeTM
(Beckman), at 4oC.
Agarose gels. For visualization of agarose gels, a Pharmacia Biotech
Electrophoresis Power Supply EPS 300 TM was used for separation of the bands of DNA
material in accordance with its instructions for use. The bands of DNA were visualized
using a FisherBiotech Transiluminator FBTIV 816TM and recorded with a Kodak TM 120 digital camera with a UV filter.
146
APPENDIX C
pET28 FABN DATA
rFab5 Heavy Chain amino acid sequence
FSLNIHPMEEEDTAMYFCQQSKEVPLTFGAGTKVELKRADAAPTVSIFPPSSEQLTSGG
ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYER
HNSYTCEATHKTSTSPIVKSFNMNHH#
rFab5 Heavy Chain DNA sequence
ATTCAGTCTCAACATCCATCCTATGGAGGAGGAAGATACTGCAATGTATTTCTGTCAGC
AAAGTAAGGAGGTTCCGCTCACGTTCGGTGCTGGGACCAAGGTGGAGCTGAAACGGGCT
GATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGG
TGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGA
AGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGC
AAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACG
ACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGA
GCTTCAACATGAATCACCACTAA
147
pET28-Fab3
TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGC
GCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCT
TCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTT
AGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATG
GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCC
ACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGT
CTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGC
TGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGT
GGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTC
AAATATGTATCCGCTCATGAATTAATTCTTAGAAAATGCTCGAGACTCATCGAGCATCA
AATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGT
TTCTGTAATGCGGCCGCAGCTAGCCTCACCGAGGCAGTTCCATAGGATGGCAAGATCCT
GGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCG
TCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAA
TGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGT
CATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGA
CGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCG
CAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATA
CCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTA
CGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGAC
CATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTG
GCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCG
148
CGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGA
GCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAG
CAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGC
GTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAA
TCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAA
GAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC
TGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA
CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT
CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAAC
GGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACC
TACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT
CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC
CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGT
GATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGG
TTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTC
TGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGA
CCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTC
CTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGC
TCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATG
GCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCC
GGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTT
CACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGA
AGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAG
CGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGG
149
TCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAA
CGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACG
TTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGG
GTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCAT
CCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACT
TTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGC
AGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGG
CAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGG
GGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAG
TGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATC
ATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCAC
CTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGC
CCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGAT
CCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTT
CCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAG
GCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAG
CTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTT
GCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTG
TCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTC
GGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGG
GAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAG
TCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCC
AGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCT
GGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAA
150
ATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGT
GCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCC
CACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTT
CGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGC
CGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCA
ACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCC
ATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCAC
GCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTG
GTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGA
AAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCT
GCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAAT
GGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACC
CACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGA
TGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATG
CGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGG
GAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGA
GATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGC
GGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCGAATTCGAG
CTCATGAAAAAGCAGCTTTCGCGATTTGCAGTGGCACTGCTGGTTTCGCTACCGTGGCC
CAGGCGGGCCGAGCTCGTGTTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGC
AGAGGGCCACCATCTCNTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTT
ATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATC
CAACCTAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCA
GTCTCAACATCCATCCTATGGAGGAGGAAGATACTGCAATGTATTTCTGTCAGCAAAGT
151
AAGGAGGTTCCGCTCACGTTCGGTGCTGGGACCAAGGTGGAGCTGAAACGGGCTGATGC
TGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCT
CAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATT
GATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGA
CAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATA
ACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTC
AACATGAATCACCACTAATCTAGATAACCATGGGCGTCGACAAGCTTGCGGCCGCACTC
GAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGA
GTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGG
TCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATGAAGGAGAAAACTCGAG
ATGATCGATCAGGTGCAGCTCGAGCAGCCTGGGTCTGTGCTGGTAAGGCCTGGAGCTTC
AGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTCCTGGATACACTGGG
CGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTCATCCTAATAGTGGT
AATACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACACATCCTC
CAGCACAGCCTACGTGGATCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACT
GTGCAAGATGGAGGTACGGTAGTCCCTACTACTTTGACTACTGGGGCCAAGGCACCACT
CTCACTGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATC
TGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTG
AGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCA
GCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCAC
CTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGG
ACAAGAAAATTGTGCCCCATCATACTAGTTAA
152
Open reading frame (5196-5918)
MKKQLSRFAVALLVSLPWPRRAELVLTQSPASLAVSLGQRATISCRASESVDNY
GISFMNWFQQKPGQPPKLLIYAASNLGSGVPARFSGSGSGTDFSLNIHPMEEEDT
AMYFCQQSKEVPLTFGAGTKVELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNN
FYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSY
TCEATHKTSTSPIVKSFNMNHH#
Open reading frame (6137-6814)
MIDQVQLEQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGEI
HPNSGNTNYNEKFKGKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARWRYGSPY
YFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTV
TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVD
KKIVPHHTS#
153
APPENDIX D
PHAGEMID GENEBANK ACQUISITION DATA
LOCUS pCOMBFab4 4681 bp DNA linear ROD 11-MAR-2003
DEFINITION [gene=fab4lc][source=2611..3333][gene=fab4hc][source=3363..4649]
4681 bases, 262E checksum.
ACCESSION pCOMBFab4
VERSION
KEYWORDS .
SOURCE Mus musculus (house mouse)
ORGANISM Mus musculus
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Rodentia; Sciurognathi; Muridae; Murinae; Mus.
REFERENCE 1 (bases 1 to 4681)
AUTHORS Kelley, L.-L.Clancy. and Momany, C.
TITLE Generation of a Phagemid Mouse Recombinant Antibody Fragment
Library by Multi-Site Directed Mutagenesis
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 4681)
AUTHORS Kelley, L.-L. Clancy and Momany, C.
154
TITLE Direct Submission
JOURNAL Submitted (11-MAR-2003) Department of Pharmaceutical and Biomedical
Sciences, University of Georgia, D. W. Brooks Drive, Athens, GA
30602, USA
FEATURES Location/Qualifiers
source 1..4681
/organism="Mus musculus"
/lab_host="Escherichia coli"
/plasmid="pCOMB3H"
gene 2611..3333
/gene="fab4lc"
CDS 2611..3333
/gene="fab4lc"
/codon_start=1
/product="Fab4LC"
/translation="MKKTAIAIAVALAGFATVAQAAELVLTQSPASLAVSLGQRATIS
CRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNLGSGVPARFSGSGSGTDFSL
NIHPMEEEDTAMYFCQQSKEVPLTFGAGTKVELKRADAAPTVSIFPPSSEQLTSG
GASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT
KDEYERHNSYTCEATHKTSTSPIVKSFNMNHH"
gene 3363..4649
/gene="fab4hc"
155
CDS 3363..4649
/gene="fab4hc"
/codon_start=1
/product="Fab4HC"
/translation="MKYLLPTAAAGLLLLAAQPAMAEVQLLEQPGSVLVRPGASVKLS
CKASGYTFTSSWIHWAKQRPGQGLEWIGEIHPNSGNTNYNEKFKGKATLTVDTS
SSTAYVDLSSLTSEDSAVYYCARWRYGSPYYFDYWGQGTTLTVSSAKTTPPSVY
PLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT
LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPHHTSGQAGQEGGGSEGGGS
EGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSV
ATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQ
YLPSLPQSVECRPFVFSAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANI
LRNKES"
BASE COUNT 1187 a 1151 c 1187 g 1155 t 1 others
ORIGIN
1 gggaaattgt aagcgttaata` attttgttaa aattcgcgtt aaatttttgt taaatcagct
61 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
121 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
181 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
241 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
301 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
361 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
156
421 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
481 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
541 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
601 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
661 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
721 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
781 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
841 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
901 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
961 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
1021 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
1081 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
1141 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
1201 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
1261 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
1321 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
1381 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
1441 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
1501 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
1561 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
1621 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
1681 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
1741 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
157
1801 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
1861 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
1921 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
1981 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
2041 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
2101 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
2161 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
2221 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
2281 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
2341 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
2401 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
2461 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
2521 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
2581 gataacaatt gaattcagga ggaatttaaa atgaaaaaga cagctatcgc gattgcagtg
2641 gcactggctg gtttcgctac cgtggcccag gcggccgagc tcgtgttgac ccaatctcca
2701 gcttctttgg ctgtgtctct agggcagagg gccaccatct cntgcagagc cagcgaaagt
2761 gttgataatt atggcattag ttttatgaac tggttccaac agaaaccagg acagccaccc
2821 aaactcctca tctatgctgc atccaaccta ggatccgggg tccctgccag gtttagtggc
2881 agtgggtctg ggacagactt cagtctcaac atccatccta tggaggagga agatactgca
2941 atgtatttct gtcagcaaag taaggaggtt ccgctcacgt tcggtgctgg gaccaaggtg
3001 gagctgaaac gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag
3061 ttaacatctg gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc caaagacatc
3121 aatgtcaagt ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact
158
3181 gatcaggaca gcaaagacag cacctacagc atgagcagca ccctcacgtt gaccaaggac
3241 gagtatgaac gacataacag ctatacctgt gaggccactc acaagacatc aacttcaccc
3301 attgtcaaga gcttcaacat gaatcaccac taatctagat aattaattag gaggaattta
3361 aaatgaaata cctattgcct acggcagccg ctggattgtt attactcgct gcccaaccag
3421 ccatggccga ggtgcagctg ctcgagcagc ctgggtctgt gctggtaagg cctggagctt
3481 cagtgaagct gtcctgcaag gcttctggct acaccttcac cagctcctgg atacactggg
3541 cgaagcagag gcctggacaa ggccttgagt ggattggaga gattcatcct aatagtggta
3601 atactaacta caatgagaag ttcaagggca aggccacact gactgtagac acatcctcca
3661 gcacagccta cgtggatctc agcagcctga catctgagga ctctgcggtc tattactgtg
3721 caagatggag gtacggtagt ccctactact ttgactactg gggccaaggc accactctca
3781 ctgtctcctc agccaaaacg acacccccat ctgtctatcc actggcccct ggatctgctg
3841 cccaaactaa ctccatggtg accctgggat gcctggtcaa gggctatttc cctgagccag
3901 tgacagtgac ctggaactct ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc
3961 tgcagtctga cctctacact ctgagcagct cagtgactgt cccctccagc acctggccca
4021 gcgagaccgt cacctgcaac gttgcccacc cggccagcag caccaaggtg gacaagaaaa
4081 ttgtgcccca tcatactagt ggccaggccg gccaggaggg tggtggctct gagggtggcg
4141 gttctgaggg tggcggctct gagggaggcg gttccggtgg tggctctggt tccggtgatt
4201 ttgattatga aaagatggca aacgctaata agggggctat gaccgaaaat gccgatgaaa
4261 acgcgctaca gtctgacgct aaaggcaaac ttgattctgt cgctactgat tacggtgctg
4321 ctatcgatgg tttcattggt gacgtttccg gccttgctaa tggtaatggt gctactggtg
4381 attttgctgg ctctaattcc caaatggctc aagtcggtga cggtgataat tcacctttaa
4441 tgaataattt ccgtcaatat ttaccttccc tccctcaatc ggttgaatgt cgcccttttg
4501 tctttagcgc tggtaaacca tatgaatttt ctattgattg tgacaaaata aacttattcc
159
4561 gtggtgtctt tgcgtttctt ttatatgttg ccacctttat gtatgtattt tctacgtttg
4621 ctaacatact gcgtaataag gagtcttaag ctagctaatt aatttaagcg gccgcagatc
4681 t
160